LOCUS       V022364                 8720 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V022364
VERSION     V022364
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"
     CDS             2827..3921
                     /label="WNT5A(NM_001377271)"
                     /note="WNT5A(NM_001377271)"
                     /gene="WNT5A"
     CDS             3943..3966
                     /codon_start=1
                     /product="FLAG® epitope tag, followed by an enterokinase
                     cleavage site"
                     /transl_table=1
                     /translation="DYKDDDDK"
                     /label="FLAG"
     CDS             3976..4029
                     /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             4060..4767
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /transl_table=1
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK*"
                     /label="mCherry"
     misc_feature    4774..5362
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             5436..6069
                     /note="3' long terminal repeat (LTR) from HIV-1"
                     /label="3' LTR"
     primer_bind     complement(6198..6214)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6222..6238
                     /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(6246..6276)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6291..6312
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6600..7188)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7359..8219)
                     /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(8220..8324)
                     /gene="bla"
                     /label="AmpR promoter"
     polyA_signal    8372..8506
                     /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 ctggaagtgc aatgtcttcc aagttcttcc tagtggcttt ggccatattt
     2881 ttctccttcg cccaggttgt aattgaagcc aattcttggt ggtcgctagg tatgaataac
     2941 cctgttcaga tgtcagaagt atatattata ggagcacagc ctctctgcag ccaactggca
     3001 ggactttctc aaggacagaa gaaactgtgc cacttgtatc aggaccacat gcagtacatc
     3061 ggagaaggcg cgaagacagg catcaaagaa tgccagtatc aattccgaca tcgaaggtgg
     3121 aactgcagca ctgtggataa cacctctgtt tttggcaggg tgatgcagat aggcagccgc
     3181 gagacggcct tcacatacgc ggtgagcgca gcaggggtgg tgaacgccat gagccgggcg
     3241 tgccgcgagg gcgagctgtc cacctgcggc tgcagccgcg ccgcgcgccc caaggacctg
     3301 ccgcgggact ggctctgggg cggctgcggc gacaacatcg actatggcta ccgctttgcc
     3361 aaggagttcg tggacgcccg cgagcgggag cgcatccacg ccaagggctc ctacgagagt
     3421 gctcgcatcc tcatgaacct gcacaacaac gaggccggcc gcaggacggt gtacaacctg
     3481 gctgatgtgg cctgcaagtg ccatggggtg tccggctcat gtagcctgaa gacatgctgg
     3541 ctgcagctgg cagacttccg caaggtgggt gatgccctga aggagaagta cgacagcgcg
     3601 gcggccatgc ggctcaacag ccggggcaag ttggtacagg tcaacagccg cttcaactcg
     3661 cccaccacac aagacctggt ctacatcgac cccagccctg actactgcgt gcgcaatgag
     3721 agcaccggct cgctgggcac gcagggccgc ctgtgcaaca agacgtcgga gggcatggat
     3781 ggctgcgagc tcatgtgctg cggccgtggc tacgaccagt tcaagaccgt gcagacggag
     3841 cgctgccact gcaagttcca ctggtgctgc tacgtcaagt gcaagaagtg cacggagatc
     3901 gtggaccagt ttgtgtgcaa ggaattcgga ggagggggaa gcgactacaa ggacgacgat
     3961 gacaagggca gcggtgaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat
     4021 cccggccctt ccggaatgga gagcgacgag agcggcctgg tgagcaaggg cgaggaggat
     4081 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     4141 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     4201 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     4261 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     4321 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     4381 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     4441 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     4501 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     4561 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     4621 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     4681 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     4741 ggcggcatgg acgagctgta caagtaggtc gacaatcaac ctctggatta caaaatttgt
     4801 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     4861 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     4921 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     4981 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     5041 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     5101 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     5161 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     5221 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     5281 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     5341 tccctttggg ccgcctcccc gcctggtacc tttaagacca atgacttaca aggcagctgt
     5401 agatcttagc cactttttaa aagaaaagag gggactggaa gggctaattc actcccaacg
     5461 aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattagca
     5521 gaactacaca ccagggccag gggtcagata tccactgacc tttggatggt gctacaagct
     5581 agtaccagtt gagccagata aggtagaaga ggccaataaa ggagagaaca ccagcttgtt
     5641 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtgttag agtggaggtt
     5701 tgacagccgc ctagcatttc atcacgtggc ccgagagctg catccggagt acttcaagaa
     5761 ctgctgatat cgagcttgct acaagggact ttccgctggg gactttccag ggaggcgtgg
     5821 cctgggcggg actggggagt ggcgagccct cagatcctgc atataagcag ctgctttttg
     5881 cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     5941 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     6001 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     6061 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     6121 gaatatcaga gagtgagagg ccttgacatt gctagcgttt taccgtcgac ctctagctag
     6181 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     6241 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     6301 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6361 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6421 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6481 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6541 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6601 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6661 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6721 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6781 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6841 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6901 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6961 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     7021 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     7081 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     7141 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     7201 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     7261 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     7321 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     7381 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     7441 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7501 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7561 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7621 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7681 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7741 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7801 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7861 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     7921 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7981 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     8041 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     8101 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     8161 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     8221 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     8281 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     8341 gtgccacctg acgtcgacgg atcgggagat caacttgttt attgcagctt ataatggtta
     8401 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     8461 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaact ggataactca
     8521 agctaaccaa aatcatccca aacttcccac cccataccct attaccactg ccaattacct
     8581 gtggtttcat ttactctaaa cctgtgattc ctctgaatta ttttcatttt aaagaaattg
     8641 tatttgttaa atatgtacta caaacttagt agtttttaaa gaaattgtat ttgttaaata
     8701 tgtactacaa acttagtagt
//
