LOCUS       V025082                 6887 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V025082
VERSION     V025082
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..3096
                     /label="HRH3(NM_007232)"
                     /note="HRH3(NM_007232)"
                     /gene="HRH3"
     CDS             3103..3123
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /transl_table=1
                     /translation="ENLYFQS"
                     /label="TEV site"
     CDS             3124..3837
                     /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             3838..3861
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin®, an engineered
                     form of streptavidin"
                     /transl_table=1
                     /translation="WSHPQFEK"
                     /label="Strep-Tag II"
     CDS             3880..3903
                     /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    3975..4030
                     /note="rabbit&nbsp;β-globin polyadenylation signal (Gil and
                     Proudfoot, 1987)"
                     /label="β-globin poly(A) signal"
     primer_bind     complement(4390..4406)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    4414..4430
                     /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(4438..4468)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    4483..4504
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     promoter        4563..4758
                     /note="SV40 early promoter"
                     /label="SV40 promoter"
     polyA_signal    4764..4898
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      complement(5137..5725)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(5896..6756)
                     /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(6757..6861)
                     /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 gatggagcgc gcgccgcccg acgggccgct gaacgcttcg
     1801 ggggcgctgg cgggcgaggc ggcggcggcg ggcggggcgc gcggcttctc ggcagcctgg
     1861 accgcggtgc tggccgcgct catggcgctg ctcatcgtgg ccacggtgct gggcaacgcg
     1921 ctggtcatgc tcgccttcgt ggccgactcg agcctccgca cccagaacaa cttcttcctg
     1981 ctcaacctcg ccatctccga cttcctcgtc ggcgccttct gcatcccact gtatgtaccc
     2041 tacgtgctga caggccgctg gaccttcggc cggggcctct gcaagctgtg gctggtagtg
     2101 gactacctgc tgtgcacctc ctctgccttc aacatcgtgc tcatcagcta cgaccgcttc
     2161 ctgtcggtca cccgagcggt ctcataccgg gcccagcagg gtgacacgcg gcgggcagtg
     2221 cggaagatgc tgctggtgtg ggtgctggcc ttcctgctgt acggaccagc catcctgagc
     2281 tgggagtacc tgtccggggg cagctccatc cccgagggcc actgctatgc cgagttcttc
     2341 tacaactggt acttcctcat cacggcttcc accctggagt tctttacgcc cttcctcagc
     2401 gtcaccttct ttaacctcag catctacctg aacatccaga ggcgcacccg cctccggctg
     2461 gatggggctc gagaggcagc cggccccgag ccccctcccg aggcccagcc ctcaccaccc
     2521 ccaccgcctg gctgctgggg ctgctggcag aaggggcacg gggaggccat gccgctgcac
     2581 aggtatgggg tgggtgaggc ggccgtaggc gctgaggccg gggaggcgac cctcgggggt
     2641 ggcggtgggg gcggctccgt ggcttcaccc acctccagct ccggcagctc ctcgaggggc
     2701 actgagaggc cgcgctcact caagaggggc tccaagccgt cggcgtcctc ggcctcgctg
     2761 gagaagcgca tgaagatggt gtcccagagc ttcacccagc gctttcggct gtctcgggac
     2821 aggaaagtgg ccaagtcgct ggccgtcatc gtgagcatct ttgggctctg ctgggcccca
     2881 tacacgctgc tgatgatcat ccgggccgcc tgccatggcc actgcgtccc tgactactgg
     2941 tacgaaacct ccttctggct cctgtgggcc aactcggctg tcaaccctgt cctctaccct
     3001 ctgtgccacc acagcttccg ccgggccttc accaagctgc tctgccccca gaagctcaaa
     3061 atccagcccc acagctccct ggagcactgc tggaaggaat tcgaaaacct gtacttccaa
     3121 tccgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     3181 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     3241 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     3301 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     3361 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     3421 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     3481 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     3541 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     3601 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     3661 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     3721 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     3781 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtgg
     3841 agccacccgc agttcgagaa gggcggcggc agcagcgcct ggagccaccc gcagttcgag
     3901 aagtgaagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca
     3961 tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt
     4021 gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta
     4081 tttggtttag agtttggcaa catatgccca tatgctggct gccatgaaca aaggtggcta
     4141 taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa
     4201 gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca
     4261 tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc
     4321 ccagtcatag ctgtccctct tctcttatga agatccctcg acctgcagcc caagcttggc
     4381 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4441 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4501 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat
     4561 ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4621 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4681 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4741 ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa
     4801 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4861 tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca
     4921 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4981 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5041 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5101 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5161 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5221 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5281 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     5341 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5401 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5461 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5521 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     5581 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5641 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5701 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5761 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5821 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5881 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5941 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6001 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6061 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6121 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6181 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6241 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6301 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6361 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6421 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6481 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6541 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6601 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6661 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6721 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6781 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6841 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
