LOCUS       V024829                 6968 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V024829
VERSION     V024829
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        4..383
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        386..661
                     /label="chicken β-actin promoter"
     intron          662..1674
                     /note="chimera between introns from chicken β-actin and
                     rabbit β-globin"
                     /label="chimeric intron"
     regulatory      1681..1690
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     CDS             1690..1755
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     CDS             1762..3177
                     /label="CNR1(NM_016083)"
                     /note="CNR1(NM_016083)"
                     /gene="CNR1"
     CDS             3184..3204
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /transl_table=1
                     /translation="ENLYFQS"
                     /label="TEV site"
     CDS             3205..3918
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /transl_table=1
                     /translation="V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
                     /label="EGFP"
     CDS             3919..3942
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin®, an engineered
                     form of streptavidin"
                     /transl_table=1
                     /translation="WSHPQFEK"
                     /label="Strep-Tag II"
     CDS             3961..3984
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin®, an engineered
                     form of streptavidin"
                     /transl_table=1
                     /translation="WSHPQFEK"
                     /label="Strep-Tag II"
     polyA_signal    4056..4111
                     /note="rabbit&nbsp;β-globin polyadenylation signal (Gil and
                     Proudfoot, 1987)"
                     /label="β-globin poly(A) signal"
     primer_bind     complement(4471..4487)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    4495..4511
                     /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(4519..4549)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    4564..4585
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     promoter        4644..4839
                     /note="SV40 early promoter"
                     /label="SV40 promoter"
     polyA_signal    4845..4979
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      complement(5218..5806)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(5977..6837)
                     /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(6838..6942)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gtgcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 gccaccatgg actacaaaga ccatgacggt gattataaag atcatgacat cgactacaag
     1741 gatgacgatg acaagctcga gatgaagtcg atcctagatg gccttgcaga taccaccttc
     1801 cgcaccatca ccactgacct cctgtacgtg ggctcaaatg acattcagta cgaagacatc
     1861 aaaggtgaca tggcatccaa attagggtac ttcccacaga aattcccttt aacttccttt
     1921 aggggaagtc ccttccaaga gaagatgact gcgggagaca acccccagct agtcccagca
     1981 gaccaggtga acattacaga attttacaac aagtctctct cgtccttcaa ggagaatgag
     2041 gagaacatcc agtgtgggga gaacttcatg gacatagagt gtttcatggt cctgaacccc
     2101 agccagcagc tggccattgc agtcctgtcc ctcacgctgg gcaccttcac ggtcctggag
     2161 aacctcctgg tgctgtgcgt catcctccac tcccgcagcc tccgctgcag gccttcctac
     2221 cacttcatcg gcagcctggc ggtggcagac ctcctgggga gtgtcatttt tgtctacagc
     2281 ttcattgact tccacgtgtt ccaccgcaaa gatagccgca acgtgtttct gttcaaactg
     2341 ggtggggtca cggcctcctt cactgcctcc gtgggcagcc tgttcctcac agccatcgac
     2401 aggtacatat ccattcacag gcccctggcc tataagagga ttgtcaccag gcccaaggcc
     2461 gtggtggcgt tttgcctgat gtggaccata gccattgtga tcgccgtgct gcctctcctg
     2521 ggctggaact gcgagaaact gcaatctgtt tgctcagaca ttttcccaca cattgatgaa
     2581 acctacctga tgttctggat cggggtcacc agcgtactgc ttctgttcat cgtgtatgcg
     2641 tacatgtata ttctctggaa ggctcacagc cacgccgtcc gcatgattca gcgtggcacc
     2701 cagaagagca tcatcatcca cacgtctgag gatgggaagg tacaggtgac ccggccagac
     2761 caagcccgca tggacattag gttagccaag accctggtcc tgatcctggt ggtgttgatc
     2821 atctgctggg gccctctgct tgcaatcatg gtgtatgatg tctttgggaa gatgaacaag
     2881 ctcattaaga cggtgtttgc attctgcagt atgctctgcc tgctgaactc caccgtgaac
     2941 cccatcatct atgctctgag gagtaaggac ctgcgacacg ctttccggag catgtttccc
     3001 tcttgtgaag gcactgcgca gcctctggat aacagcatgg gggactcgga ctgcctgcac
     3061 aaacacgcaa acaatgcagc cagtgttcac agggccgcag aaagctgcat caagagcacg
     3121 gtcaagattg ccaaggtaac catgtctgtg tccacagaca cgtctgccga ggctctggaa
     3181 ttcgaaaacc tgtacttcca atccgtgagc aagggcgagg agctgttcac cggggtggtg
     3241 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     3301 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     3361 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     3421 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     3481 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     3541 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     3601 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     3661 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     3721 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     3781 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     3841 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     3901 atggacgagc tgtacaagtg gagccacccg cagttcgaga agggcggcgg cagcagcgcc
     3961 tggagccacc cgcagttcga gaagtgaaga tctttttccc tctgccaaaa attatgggga
     4021 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     4081 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     4141 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
     4201 tgccatgaac aaaggtggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc
     4261 cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt
     4321 gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact agccagattt
     4381 ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg aagatccctc
     4441 gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4501 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4561 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4621 aaacctgtcg tgccagcgga tccgcatctc aattagtcag caaccatagt cccgccccta
     4681 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4741 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4801 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag
     4861 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4921 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc
     4981 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5041 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5101 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5161 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5221 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5281 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5341 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5401 ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5461 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5521 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5581 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5641 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5701 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5761 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5821 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5881 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5941 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6001 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6061 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6121 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6181 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6241 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6301 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6361 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6421 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6481 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6541 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6601 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6661 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6721 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6781 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6841 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6901 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6961 gccacctg
//
