LOCUS       V033899                 8854 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V033899
VERSION     V033899
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     LTR             234..414
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /label="5' LTR (truncated)"
     misc_feature    458..583
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
                     /label="HIV-1 Ψ"
     misc_feature    1076..1309
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
                     /label="RRE"
     CDS             1493..1537
                     /codon_start=1
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /product="antigenic peptide corresponding to amino acids
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
                     et al., 2013)"
                     /transl_table=1
                     /translation="KNEQELLELDKWASL"
                     /label="gp41 peptide"
     misc_feature    1804..1921
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
                     /label="cPPT/CTS"
     promoter        2011..2214
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     CDS             2317..2880
                     /label="Kras(NM_021284)"
                     /note="Kras(NM_021284)"
                     /gene="Kras"
     CDS             2887..2952
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     promoter        2996..3207
                     /note="core&nbsp;promoter for human elongation factor
                     EF-1α"
                     /label="EF-1α core promoter"
     LTR             3220..3488
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
                     /label="5' LTR (truncated)"
     regulatory      3517..3526
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     CDS             3547..4209
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /transl_table=1
                     /translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
                     FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
                     EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
                     YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
                     /label="CopGFP"
     CDS             4279..4332
                     /codon_start=1
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /product="2A peptide from&nbsp;Thosea asigna virus capsid
                     protein"
                     /protein_id=""
                     /transl_table=1
                     /translation="EGRGSLLTCGDVEENPGP"
                     /label="T2A"
     CDS             4333..4932
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /note="confers resistance to puromycin"
                     /product="puromycin N-acetyltransferase"
                     /transl_table=1
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
                     /label="PuroR"
     misc_feature    4933..5521
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             5595..5828
                     /note="self-inactivating&nbsp;3' long terminal repeat (LTR)
                     from HIV-1"
                     /label="3' LTR (ΔU3)"
     polyA_signal    5900..6021
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      6061..6196
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     complement(6229..6245)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6253..6269
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-β-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     promoter        complement(6277..6307)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6322..6343
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6631..7219)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7390..8250)
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     promoter        complement(8251..8355)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     8829..8845
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 fwd"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc
      241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
      421 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct
      481 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
      541 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
      601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
      661 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
      721 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
      781 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
      841 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
      901 taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg
      961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1081 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc
     1141 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1201 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1261 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1381 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt
     1801 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     1861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     1921 tatcgatact agtattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     1981 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     2041 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     2101 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     2161 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggttt atataagcag agctcgttta
     2221 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagattc
     2281 tagagctagc ctgatacgaa ctcggaattc gccaccatga ctgagtataa acttgtggtg
     2341 gttggagctg atggcgtagg caagagcgcc ttgacgatac agctaattca gaatcacttt
     2401 gtggatgagt atgaccctac gatagaggac tcctacagga aacaagtagt aattgatgga
     2461 gaaacctgtc tcttggatat tctcgacaca gcaggtcaag aggagtacag tgcaatgagg
     2521 gaccagtaca tgagaactgg ggagggcttt ctttgtgtat ttgccataaa taatactaaa
     2581 tcatttgaag atattcacca ttatagagaa caaattaaaa gagtaaagga ctctgaagat
     2641 gtgcctatgg tcctggtagg gaataagtgt gatttgcctt ctagaacagt agacacgaaa
     2701 caggctcagg agttagcaag gagttacggg attccgttca ttgagacctc agcaaagaca
     2761 agacagggtg ttgacgatgc cttctataca ttagtccgag aaattcgaaa acataaagaa
     2821 aagatgagca aagatgggaa gaagaagaag aagaagtcaa ggacaaggtg tacagttatg
     2881 ggatccgact acaaagacca tgacggtgat tataaagatc atgacatcga ctacaaggat
     2941 gacgatgaca agtagcggcc gcgaaggatc tgcgatcgct ccggtgcccg tcagtgggca
     3001 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt
     3061 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     3121 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     3181 cgcaacgggt ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca
     3241 cgcgcccgcc gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct
     3301 cccgcctgtg gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg
     3361 agaccgggcc tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg
     3421 ctttgcctga ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt
     3481 tacagatcca agctgtgacc ggcgcctacg ctagacgcca ccatggagag cgacgagagc
     3541 ggcctgcccg ccatggagat cgagtgccgc atcaccggca ccctgaacgg cgtggagttc
     3601 gagctggtgg gcggcggaga gggcaccccc aagcagggcc gcatgaccaa caagatgaag
     3661 agcaccaaag gcgccctgac cttcagcccc tacctgctga gccacgtgat gggctacggc
     3721 ttctaccact tcggcaccta ccccagcggc tacgagaacc ccttcctgca cgccatcaac
     3781 aacggcggct acaccaacac ccgcatcgag aagtacgagg acggcggcgt gctgcacgtg
     3841 agcttcagct accgctacga ggccggccgc gtgatcggcg acttcaaggt ggtgggcacc
     3901 ggcttccccg aggacagcgt gatcttcacc gacaagatca tccgcagcaa cgccaccgtg
     3961 gagcacctgc accccatggg cgataacgtg ctggtgggca gcttcgcccg caccttcagc
     4021 ctgcgcgacg gcggctacta cagcttcgtg gtggacagcc acatgcactt caagagcgcc
     4081 atccacccca gcatcctgca gaacgggggc cccatgttcg ccttccgccg cgtggaggag
     4141 ctgcacagca acaccgagct gggcatcgtg gagtaccagc acgccttcaa gacccccatc
     4201 gccttcgcca gatcccgcgc tcagtcgtcc aattctgccg tggacggcac cgccggaccc
     4261 ggctccaccg gatctcgcga gggcagagga agtcttctaa catgcggtga cgtggaggag
     4321 aatcccggcc ctatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     4381 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     4441 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     4501 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     4561 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     4621 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     4681 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     4741 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     4801 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     4861 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     4921 cccggtgcct gaaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     4981 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     5041 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     5101 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     5161 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     5221 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     5281 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     5341 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     5401 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     5461 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     5521 cctggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     5581 agaaaagggg ggactggaag ggctaattca ctcccaacga agataagatc tgctttttgc
     5641 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     5701 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     5761 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     5821 ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     5881 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     5941 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     6001 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     6061 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     6121 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     6181 ggcttttttg gaggcctaga cttttgcaga gaccaaattc gtaatcatgt catagctgtt
     6241 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6301 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6361 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6421 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6481 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6541 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6601 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6661 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6721 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6781 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6841 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6901 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6961 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     7021 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     7081 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     7141 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     7201 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     7261 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     7321 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7381 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7441 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7501 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7561 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7621 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7681 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7741 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7801 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7861 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7921 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7981 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     8041 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     8101 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8161 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8221 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     8281 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     8341 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     8401 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     8461 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     8521 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     8581 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     8641 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
     8701 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     8761 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     8821 acgacgttgt aaaacgacgg ccagtgccaa gctg
//
