LOCUS       V041158                 9531 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V041158
VERSION     V041158
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     LTR             1..634
                     /note="3' long terminal repeat (LTR) from HIV-1"
                     /label="3' LTR"
     misc_feature    681..806
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
                     /label="HIV-1 Ψ"
     misc_feature    1303..1536
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
                     /label="RRE"
     CDS             1721..1765
                     /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    2028..2143
                     /note="central polypurine tract and central termination
                     sequence of HIV-1 (lacking the first T)"
                     /label="cPPT/CTS"
     enhancer        2201..2504
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        2505..2708
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     regulatory      2821..2830
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     CDS             2833..2922
                     /codon_start=1
                     /product="two Strep-Tag®II moieties connected by a linker
                     for enhanced binding to Strep-Tactin®,&nbsp;an engineered
                     form of streptavidin (Schmidt et al., 2013)"
                     /transl_table=1
                     /translation="SA,WSHPQFEK,GGGSGGGSGGSA,WSHPQFEK"
                     /label="Twin-Strep-tag"
     CDS             2938..3588
                     /label="Grb2(NM_008163)"
                     /note="Grb2(NM_008163)"
                     /gene="Grb2"
     CDS             3619..4338
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="Aequorea coerulescens GFP"
                     /transl_table=1
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK*"
                     /label="AcGFP1"
     promoter        4366..4865
                     /note="mouse phosphoglycerate kinase 1 promoter"
                     /label="PGK promoter"
     CDS             4886..5485
                     /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    5499..6087
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             6294..6927
                     /note="3' long terminal repeat (LTR) from HIV-1"
                     /label="3' LTR"
     primer_bind     complement(7056..7072)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    7080..7096
                     /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(7104..7134)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    7149..7170
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(7458..8046)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(8217..9077)
                     /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(9078..9182)
                     /gene="bla"
                     /label="AmpR promoter"
     polyA_signal    9230..9364
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2161 acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa
     2221 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2281 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2341 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2401 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2461 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2521 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     2581 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2641 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2701 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
     2761 tccatagaag acaccgactc tactagagga tcgctagcgc taccggactc agatctcgag
     2821 gccaccatgg cttcagcatg gagccatccg cagtttgaaa aaggtggtgg tagcggtggt
     2881 ggttcaggtg gtagtgcatg gtcacaccct cagtttgaga aattcgaata cgaattcatg
     2941 gaagccatcg ccaaatatga cttcaaagct actgctgacg atgagctgag cttcaaaagg
     3001 ggggacatcc ttaaggtttt gaatgaagaa tgtgaccaga actggtataa ggcagaactc
     3061 aatgggaaag atggcttcat ccccaagaat tacatagaaa tgaaaccaca tccgtggttt
     3121 tttggcaaaa tccccagagc caaggcagaa gaaatgctca gcaaacagcg gcatgacggg
     3181 gccttcctga tccgagagag cgagagcgct cctggggact tctccctgtc cgtcaagttt
     3241 ggaaatgatg tgcagcactt caaggtgctc cgcgacggag ccgggaagta tttcctgtgg
     3301 gtggtgaagt ttaattcttt gaatgagctg gtagattacc acagatcaac atccgtgtcc
     3361 aggaaccagc agatattctt acgggacata gaacagatgc cacagcagcc aacctacgtc
     3421 caggcgctct ttgactttga cccccaggag gatggcgagc tgggctttcg cagaggagac
     3481 ttcattcatg tcatggataa ctcagatccc aattggtgga aaggggcctg ccacgggcag
     3541 accggcatgt ttccccgcaa ttatgtcacc ccagtgaacc ggaacgtcgg tggaggcgga
     3601 tcaggaggcg gtggatctat ggtgagcaag ggcgccgagc tgttcaccgg catcgtgccc
     3661 atcctgatcg agctgaatgg cgatgtgaat ggccacaagt tcagcgtgag cggcgagggc
     3721 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     3781 cctgtgccct ggcccaccct ggtgaccacc ctgagctacg gcgtgcagtg cttctcacgc
     3841 taccccgatc acatgaagca gcacgacttc ttcaagagcg ccatgcctga gggctacatc
     3901 caggagcgca ccatcttctt cgaggatgac ggcaactaca agtcgcgcgc cgaggtgaag
     3961 ttcgagggcg ataccctggt gaatcgcatc gagctgaccg gcaccgattt caaggaggat
     4021 ggcaacatcc tgggcaataa gatggagtac aactacaacg cccacaatgt gtacatcatg
     4081 accgacaagg ccaagaatgg catcaaggtg aacttcaaga tccgccacaa catcgaggat
     4141 ggcagcgtgc agctggccga ccactaccag cagaataccc ccatcggcga tggccctgtg
     4201 ctgctgcccg ataaccacta cctgtccacc cagagcgccc tgtccaagga ccccaacgag
     4261 aagcgcgatc acatgatcta cttcggcttc gtgaccgccg ccgccatcac ccacggcatg
     4321 gatgagctgt acaagtgagc ggccgcgact ctagataatt ctaccgggta ggggaggcgc
     4381 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     4441 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     4501 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     4561 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     4621 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     4681 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     4741 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     4801 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     4861 tttcgacctg cagcccaagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac
     4921 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     4981 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     5041 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     5101 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     5161 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     5221 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     5281 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     5341 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     5401 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     5461 catgacccgc aagcccggtg cctgaccgcg tctggaacaa tcaacctctg gattacaaaa
     5521 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     5581 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     5641 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     5701 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     5761 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     5821 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     5881 tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc
     5941 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     6001 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     6061 ggatctccct ttgggccgcc tccccgcctg gaattaattc tgcagtcgag acctagaaaa
     6121 acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga
     6181 agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat
     6241 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaagaggg gactggaagg
     6301 gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg
     6361 ctacttccct gattagcaga actacacacc agggccaggg gtcagatatc cactgacctt
     6421 tggatggtgc tacaagctag taccagttga gccagataag gtagaagagg ccaataaagg
     6481 agagaacacc agcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga
     6541 agtgttagag tggaggtttg acagccgcct agcatttcat cacgtggccc gagagctgca
     6601 tccggagtac ttcaagaact gctgatatcg agcttgctac aagggacttt ccgctgggga
     6661 ctttccaggg aggcgtggcc tgggcgggac tggggagtgg cgagccctca gatcctgcat
     6721 ataagcagct gctttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg
     6781 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     6841 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     6901 tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt
     6961 tataacttgc aaagaaatga atatcagaga gtgagaggcc ttgacattgc tagcgtttta
     7021 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     7081 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     7141 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     7201 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     7261 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     7321 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     7381 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     7441 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     7501 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     7561 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     7621 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     7681 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     7741 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     7801 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     7861 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     7921 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     7981 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     8041 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     8101 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     8161 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     8221 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     8281 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     8341 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     8401 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     8461 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     8521 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     8581 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     8641 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     8701 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     8761 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     8821 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     8881 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     8941 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     9001 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     9061 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     9121 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     9181 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatca acttgtttat
     9241 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     9301 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     9361 gatcaactgg ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc cataccctat
     9421 taccactgcc aattacctgt ggtttcattt actctaaacc tgtgattcct ctgaattatt
     9481 ttcattttaa agaaattgta tttgttaaat atgtactaca aacttagtag t
//
