LOCUS       V035977                 8824 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V035977
VERSION     V035977
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..2850
                     /label="Cav1(NM_007616)"
                     /note="Cav1(NM_007616)"
                     /gene="Cav1"
     CDS             2857..2922
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     promoter        2966..3177
                     /note="core&nbsp;promoter for human elongation factor
                     EF-1α"
                     /label="EF-1α core promoter"
     LTR             3190..3458
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
                     /label="5' LTR (truncated)"
     regulatory      3487..3496
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     CDS             3517..4179
                     /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             4249..4302
                     /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             4303..4902
                     /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    4903..5491
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             5565..5798
                     /note="self-inactivating&nbsp;3' long terminal repeat (LTR)
                     from HIV-1"
                     /label="3' LTR (ΔU3)"
     polyA_signal    5870..5991
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      6031..6166
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     complement(6199..6215)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6223..6239
                     /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(6247..6277)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6292..6313
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6601..7189)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7360..8220)
                     /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(8221..8325)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     8799..8815
                     /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 gccaccatgt ctgggggcaa atacgtagac
     2341 tccgagggac atctctacac tgttcccatc cgggaacagg gcaacatcta caagcccaac
     2401 aacaaggcca tggcagacga ggtgactgag aagcaagtgt atgacgcgca caccaaggag
     2461 attgacctgg tcaaccgcga ccccaagcat ctcaacgacg acgtggtcaa gattgacttt
     2521 gaagatgtga ttgcagaacc agaagggaca cacagtttcg acggcatctg gaaggccagc
     2581 ttcaccacct tcactgtgac aaaatattgg ttttaccgct tgttgtctac gatcttcggc
     2641 atcccaatgg cactcatctg gggcatttac tttgccattc tctccttcct gcacatctgg
     2701 gcggttgtac cgtgcatcaa gagcttcctg attgagattc agtgcatcag ccgcgtctac
     2761 tccatctacg tccatacctt ctgcgatcca ctctttgaag ctattggcaa gatattcagc
     2821 aacatccgca tcagcacgca gaaagagata ggatccgact acaaagacca tgacggtgat
     2881 tataaagatc atgacatcga ctacaaggat gacgatgaca agtagcggcc gcgaaggatc
     2941 tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     3001 ttggggggag gggtcggcaa ttgaacgggt gcctagagaa ggtggcgcgg ggtaaactgg
     3061 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     3121 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacagctg
     3181 aagcttcgag gggctcgcat ctctccttca cgcgcccgcc gccctacctg aggccgccat
     3241 ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg gtgcctcctg aactgcgtcc
     3301 gccgtctagg taagtttaaa gctcaggtcg agaccgggcc tttgtccggc gctcccttgg
     3361 agcctaccta gactcagccg gctctccacg ctttgcctga ccctgcttgc tcaactctac
     3421 gtctttgttt cgttttctgt tctgcgccgt tacagatcca agctgtgacc ggcgcctacg
     3481 ctagacgcca ccatggagag cgacgagagc ggcctgcccg ccatggagat cgagtgccgc
     3541 atcaccggca ccctgaacgg cgtggagttc gagctggtgg gcggcggaga gggcaccccc
     3601 aagcagggcc gcatgaccaa caagatgaag agcaccaaag gcgccctgac cttcagcccc
     3661 tacctgctga gccacgtgat gggctacggc ttctaccact tcggcaccta ccccagcggc
     3721 tacgagaacc ccttcctgca cgccatcaac aacggcggct acaccaacac ccgcatcgag
     3781 aagtacgagg acggcggcgt gctgcacgtg agcttcagct accgctacga ggccggccgc
     3841 gtgatcggcg acttcaaggt ggtgggcacc ggcttccccg aggacagcgt gatcttcacc
     3901 gacaagatca tccgcagcaa cgccaccgtg gagcacctgc accccatggg cgataacgtg
     3961 ctggtgggca gcttcgcccg caccttcagc ctgcgcgacg gcggctacta cagcttcgtg
     4021 gtggacagcc acatgcactt caagagcgcc atccacccca gcatcctgca gaacgggggc
     4081 cccatgttcg ccttccgccg cgtggaggag ctgcacagca acaccgagct gggcatcgtg
     4141 gagtaccagc acgccttcaa gacccccatc gccttcgcca gatcccgcgc tcagtcgtcc
     4201 aattctgccg tggacggcac cgccggaccc ggctccaccg gatctcgcga gggcagagga
     4261 agtcttctaa catgcggtga cgtggaggag aatcccggcc ctatgaccga gtacaagccc
     4321 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     4381 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     4441 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     4501 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     4561 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     4621 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     4681 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     4741 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     4801 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     4861 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaaatcaacc tctggattac
     4921 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     4981 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     5041 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     5101 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     5161 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     5221 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     5281 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     5341 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     5401 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     5461 agtcggatct ccctttgggc cgcctccccg cctggtacct ttaagaccaa tgacttacaa
     5521 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     5581 ctcccaacga agataagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     5641 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     5701 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     5761 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat
     5821 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     5881 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5941 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     6001 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     6061 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     6121 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctaga cttttgcaga
     6181 gaccaaattc gtaatcatgt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     6241 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     6301 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6361 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6421 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6481 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6541 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6601 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6661 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6721 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6781 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6841 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6901 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6961 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7021 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     7081 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7141 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7201 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7261 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7321 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7381 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7441 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7501 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7561 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7621 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7681 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7741 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7801 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7861 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7921 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7981 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8041 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     8101 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8161 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8221 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8281 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8341 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     8401 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     8461 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     8521 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     8581 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     8641 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     8701 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     8761 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa
     8821 gctg
//
