LOCUS       V032518                 9526 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V032518
VERSION     V032518
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..3552
                     /label="Pdk4(NM_013743)"
                     /note="Pdk4(NM_013743)"
                     /gene="Pdk4"
     CDS             3559..3624
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     promoter        3668..3879
                     /note="core&nbsp;promoter for human elongation factor
                     EF-1α"
                     /label="EF-1α core promoter"
     LTR             3892..4160
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
                     /label="5' LTR (truncated)"
     regulatory      4189..4198
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     CDS             4219..4881
                     /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             4951..5004
                     /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             5005..5604
                     /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    5605..6193
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             6267..6500
                     /note="self-inactivating&nbsp;3' long terminal repeat (LTR)
                     from HIV-1"
                     /label="3' LTR (ΔU3)"
     polyA_signal    6572..6693
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      6733..6868
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     complement(6901..6917)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6925..6941
                     /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(6949..6979)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6994..7015
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(7303..7891)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(8062..8922)
                     /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(8923..9027)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     9501..9517
                     /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 aggcagcccg cttcgtgatg
     2341 cgcagcgcca gctcgctgag cagcgccagc ctggtcccca gggaggtcga gctgttctcc
     2401 cgctacagcc cgtccccgct gtccatgaag cagctgctgg actttggttc agaaaatgcc
     2461 tgtgaaagaa cgtcctttgc ttttctgcgg caagagctgc ccgtccgcct ggccaatatc
     2521 ctgaaggaga ttgacatcct gcctgaccgc ttagtgaaca ctccttcggt gcagctggtg
     2581 aagagctggt atatccagag cctgatggat ttggtggagt tccatgagaa gagcccagaa
     2641 gaccagaaag ccctgtcaga gtttgtagac acgctggtca aagttcgaaa cagacatcat
     2701 aatgtggtcc ctacaatggc tcaaggcatc ctggagtata aagacacctg cacagtggac
     2761 cccgttacca atcaaaatct tcagtatttt ttagaccggt tttacatgaa ccgcatttct
     2821 actcggatgc tcatgaatca gcacatcctc atattcagtg actcaaagac gggaaaccca
     2881 agccacattg gaagtatcga cccaaactgt gatgtggtag cagtagtcca agatgccttt
     2941 gagtgtgcaa agatgctctg cgaccagtat tatctaacat cgccagaatt aaacctcaca
     3001 caagtcaatg gaaaatttcc aggccaacca atccacattg tgtacgttcc ttcacacctt
     3061 caccacatgc tcttcgaact cttcaagaat gccatgaggg ccacggtcga gcatcaagaa
     3121 aaccgtcctt ccttgacccc agtagaggcc actgtcgtct tgggaaaaga agaccttaca
     3181 atcaagattt ctgaccgagg aggcggtgtt cctctgagga ttactgaccg cctctttagt
     3241 tacacgtact ccactgctcc aacacctgtg atggacaatt cccggaatgc ccctttggct
     3301 ggttttggtt atggcttgcc aatttctcgt ctctacgcca agtattttca aggagatctg
     3361 aatctctact ctatgtcagg ttatgggaca gacgctatca tctacttaaa ggctttatct
     3421 tctgagtctg tagaaaagct cccagtcttt aacaagtcag ccttcaaaca ttatcagatg
     3481 agctccgaag ctgatgactg gtgtatccca agcagggaac cgaagaacct ggcgaaggag
     3541 aagctggcag tgggatccga ctacaaagac catgacggtg attataaaga tcatgacatc
     3601 gactacaagg atgacgatga caagtagcgg ccgcgaagga tctgcgatcg ctccggtgcc
     3661 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     3721 aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     3781 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     3841 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg aggggctcgc
     3901 atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg gttgagtcgc
     3961 gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta
     4021 aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc tagactcagc
     4081 cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt ttcgttttct
     4141 gttctgcgcc gttacagatc caagctgtga ccggcgccta cgctagacgc caccatggag
     4201 agcgacgaga gcggcctgcc cgccatggag atcgagtgcc gcatcaccgg caccctgaac
     4261 ggcgtggagt tcgagctggt gggcggcgga gagggcaccc ccaagcaggg ccgcatgacc
     4321 aacaagatga agagcaccaa aggcgccctg accttcagcc cctacctgct gagccacgtg
     4381 atgggctacg gcttctacca cttcggcacc taccccagcg gctacgagaa ccccttcctg
     4441 cacgccatca acaacggcgg ctacaccaac acccgcatcg agaagtacga ggacggcggc
     4501 gtgctgcacg tgagcttcag ctaccgctac gaggccggcc gcgtgatcgg cgacttcaag
     4561 gtggtgggca ccggcttccc cgaggacagc gtgatcttca ccgacaagat catccgcagc
     4621 aacgccaccg tggagcacct gcaccccatg ggcgataacg tgctggtggg cagcttcgcc
     4681 cgcaccttca gcctgcgcga cggcggctac tacagcttcg tggtggacag ccacatgcac
     4741 ttcaagagcg ccatccaccc cagcatcctg cagaacgggg gccccatgtt cgccttccgc
     4801 cgcgtggagg agctgcacag caacaccgag ctgggcatcg tggagtacca gcacgccttc
     4861 aagaccccca tcgccttcgc cagatcccgc gctcagtcgt ccaattctgc cgtggacggc
     4921 accgccggac ccggctccac cggatctcgc gagggcagag gaagtcttct aacatgcggt
     4981 gacgtggagg agaatcccgg ccctatgacc gagtacaagc ccacggtgcg cctcgccacc
     5041 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     5101 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     5161 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     5221 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     5281 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     5341 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     5401 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     5461 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     5521 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     5581 atgacccgca agcccggtgc ctgaaatcaa cctctggatt acaaaatttg tgaaagattg
     5641 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     5701 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     5761 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     5821 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     5881 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     5941 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     6001 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     6061 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     6121 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     6181 gccgcctccc cgcctggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
     6241 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagataaga
     6301 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     6361 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     6421 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     6481 agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt atttataact
     6541 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
     6601 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     6661 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
     6721 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     6781 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     6841 aagtagtgag gaggcttttt tggaggccta gacttttgca gagaccaaat tcgtaatcat
     6901 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     6961 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     7021 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     7081 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     7141 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     7201 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     7261 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     7321 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     7381 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     7441 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     7501 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     7561 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     7621 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     7681 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     7741 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     7801 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     7861 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     7921 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     7981 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     8041 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     8101 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     8161 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     8221 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     8281 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     8341 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     8401 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     8461 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     8521 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     8581 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     8641 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     8701 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     8761 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     8821 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     8881 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     8941 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     9001 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     9061 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     9121 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     9181 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     9241 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     9301 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     9361 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     9421 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     9481 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctg
//
