LOCUS       V043713                 8770 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V043713
VERSION     V043713
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..2796
                     /label="TWIST2(NM_057179)"
                     /note="TWIST2(NM_057179)"
                     /gene="TWIST2"
     CDS             2803..2868
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     promoter        2912..3123
                     /note="core&nbsp;promoter for human elongation factor
                     EF-1α"
                     /label="EF-1α core promoter"
     LTR             3136..3404
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
                     /label="5' LTR (truncated)"
     regulatory      3433..3442
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     CDS             3463..4125
                     /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             4195..4248
                     /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             4249..4848
                     /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    4849..5437
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             5511..5744
                     /note="self-inactivating&nbsp;3' long terminal repeat (LTR)
                     from HIV-1"
                     /label="3' LTR (ΔU3)"
     polyA_signal    5816..5937
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      5977..6112
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     complement(6145..6161)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6169..6185
                     /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(6193..6223)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6238..6259
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6547..7135)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7306..8166)
                     /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(8167..8271)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     8745..8761
                     /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 gccaccatgg aggagggctc cagctcgccc
     2341 gtgtcccccg tggacagcct gggcaccagc gaggaggagc tcgagaggca gcccaagcgc
     2401 ttcggccgga agcggcgcta cagcaagaag tcgagcgaag atggcagccc gaccccgggc
     2461 aagcgcggca agaagggcag ccccagcgcg cagtccttcg aggagctgca gagccagcgc
     2521 atcctggcca acgtgcgcga gcgccagcgc acccagtcgc tcaacgaggc cttcgcggcg
     2581 ctgcgcaaga tcatccccac gctgccctct gacaagctga gcaagatcca gacgctcaag
     2641 ctggccgcca ggtacataga cttcctctac caggtcctgc agagcgacga gatggacaat
     2701 aagatgacca gctgcagcta cgtggcccac gagcgcctca gctacgcctt ctccgtgtgg
     2761 cgcatggagg gcgcgtggtc catgtccgcc tcccacggat ccgactacaa agaccatgac
     2821 ggtgattata aagatcatga catcgactac aaggatgacg atgacaagta gcggccgcga
     2881 aggatctgcg atcgctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     2941 gagaagttgg ggggaggggt cggcaattga acgggtgcct agagaaggtg gcgcggggta
     3001 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     3061 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     3121 cagctgaagc ttcgaggggc tcgcatctct ccttcacgcg cccgccgccc tacctgaggc
     3181 cgccatccac gccggttgag tcgcgttctg ccgcctcccg cctgtggtgc ctcctgaact
     3241 gcgtccgccg tctaggtaag tttaaagctc aggtcgagac cgggcctttg tccggcgctc
     3301 ccttggagcc tacctagact cagccggctc tccacgcttt gcctgaccct gcttgctcaa
     3361 ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca gatccaagct gtgaccggcg
     3421 cctacgctag acgccaccat ggagagcgac gagagcggcc tgcccgccat ggagatcgag
     3481 tgccgcatca ccggcaccct gaacggcgtg gagttcgagc tggtgggcgg cggagagggc
     3541 acccccaagc agggccgcat gaccaacaag atgaagagca ccaaaggcgc cctgaccttc
     3601 agcccctacc tgctgagcca cgtgatgggc tacggcttct accacttcgg cacctacccc
     3661 agcggctacg agaacccctt cctgcacgcc atcaacaacg gcggctacac caacacccgc
     3721 atcgagaagt acgaggacgg cggcgtgctg cacgtgagct tcagctaccg ctacgaggcc
     3781 ggccgcgtga tcggcgactt caaggtggtg ggcaccggct tccccgagga cagcgtgatc
     3841 ttcaccgaca agatcatccg cagcaacgcc accgtggagc acctgcaccc catgggcgat
     3901 aacgtgctgg tgggcagctt cgcccgcacc ttcagcctgc gcgacggcgg ctactacagc
     3961 ttcgtggtgg acagccacat gcacttcaag agcgccatcc accccagcat cctgcagaac
     4021 gggggcccca tgttcgcctt ccgccgcgtg gaggagctgc acagcaacac cgagctgggc
     4081 atcgtggagt accagcacgc cttcaagacc cccatcgcct tcgccagatc ccgcgctcag
     4141 tcgtccaatt ctgccgtgga cggcaccgcc ggacccggct ccaccggatc tcgcgagggc
     4201 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctat gaccgagtac
     4261 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     4321 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     4381 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     4441 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     4501 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     4561 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     4621 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     4681 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     4741 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     4801 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaaa tcaacctctg
     4861 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     4921 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     4981 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     5041 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     5101 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     5161 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     5221 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     5281 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     5341 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     5401 cagacgagtc ggatctccct ttgggccgcc tccccgcctg gtacctttaa gaccaatgac
     5461 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     5521 aattcactcc caacgaagat aagatctgct ttttgcttgt actgggtctc tctggttaga
     5581 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     5641 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     5701 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca
     5761 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     5821 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5881 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5941 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     6001 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     6061 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagacttt
     6121 tgcagagacc aaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct
     6181 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6241 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6301 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6361 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6421 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6481 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6541 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6601 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6661 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6721 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6781 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6841 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6901 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6961 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     7021 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     7081 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     7141 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     7201 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     7261 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     7321 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7381 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     7441 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7501 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7561 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7621 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7681 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7741 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7801 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7861 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7921 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7981 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     8041 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     8101 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     8161 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     8221 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     8281 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     8341 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     8401 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     8461 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     8521 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     8581 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
     8641 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     8701 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     8761 tgccaagctg
//
