LOCUS       V024976                 6650 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V024976
VERSION     V024976
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..2859
                     /label="GHSR(NM_198407)"
                     /note="GHSR(NM_198407)"
                     /gene="GHSR"
     CDS             2866..2886
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /transl_table=1
                     /translation="ENLYFQS"
                     /label="TEV site"
     CDS             2887..3600
                     /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             3601..3624
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin®, an engineered
                     form of streptavidin"
                     /transl_table=1
                     /translation="WSHPQFEK"
                     /label="Strep-Tag II"
     CDS             3643..3666
                     /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    3738..3793
                     /note="rabbit&nbsp;β-globin polyadenylation signal (Gil and
                     Proudfoot, 1987)"
                     /label="β-globin poly(A) signal"
     primer_bind     complement(4153..4169)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    4177..4193
                     /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(4201..4231)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    4246..4267
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     promoter        4326..4521
                     /note="SV40 early promoter"
                     /label="SV40 promoter"
     polyA_signal    4527..4661
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      complement(4900..5488)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(5659..6519)
                     /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(6520..6624)
                     /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 gatgtggaac gcgacgccca gcgaagagcc ggggttcaac
     1801 ctcacactgg ccgacctgga ctgggatgct tcccccggca acgactcgct gggcgacgag
     1861 ctgctgcagc tcttccccgc gccgctgctg gcgggcgtca cagccacctg cgtggcactc
     1921 ttcgtggtgg gcatcgctgg caacctgctc accatgctgg tggtgtcgcg cttccgcgag
     1981 ctgcgcacca ccaccaacct ctacctgtcc agcatggcct tctccgatct gctcatcttc
     2041 ctctgcatgc ccctggacct cgttcgcctc tggcagtacc ggccctggaa cttcggcgac
     2101 ctcctctgca aactcttcca attcgtcagt gagagctgca cctacgccac ggtgctcacc
     2161 atcacagcgc tgagcgtcga gcgctacttc gccatctgct tcccactccg ggccaaggtg
     2221 gtggtcacca aggggcgggt gaagctggtc atcttcgtca tctgggccgt ggccttctgc
     2281 agcgccgggc ccatcttcgt gctagtcggg gtggagcacg agaacggcac cgacccttgg
     2341 gacaccaacg agtgccgccc caccgagttt gcggtgcgct ctggactgct cacggtcatg
     2401 gtgtgggtgt ccagcatctt cttcttcctt cctgtcttct gtctcacggt cctctacagt
     2461 ctcatcggca ggaagctgtg gcggaggagg cgcggcgatg ctgtcgtggg tgcctcgctc
     2521 agggaccaga accacaagca aaccgtgaaa atgctggctg tagtggtgtt tgccttcatc
     2581 ctctgctggc tccccttcca cgtagggcga tatttatttt ccaaatcctt tgagcctggc
     2641 tccttggaga ttgctcagat cagccagtac tgcaacctcg tgtcctttgt cctcttctac
     2701 ctcagtgctg ccatcaaccc cattctgtac aacatcatgt ccaagaagta ccgggtggca
     2761 gtgttcagac ttctgggatt cgaacccttc tcccagagaa agctctccac tctgaaagat
     2821 gaaagttctc gggcctggac agaatctagt attaatacag aattcgaaaa cctgtacttc
     2881 caatccgtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2941 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3001 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3061 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     3121 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     3181 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3241 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     3301 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     3361 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     3421 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     3481 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     3541 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3601 tggagccacc cgcagttcga gaagggcggc ggcagcagcg cctggagcca cccgcagttc
     3661 gagaagtgaa gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga
     3721 gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt
     3781 tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga
     3841 gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggtgg
     3901 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga
     3961 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta
     4021 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta
     4081 ctcccagtca tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt
     4141 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4201 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4261 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg
     4321 gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4381 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4441 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4501 ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat
     4561 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4621 gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg
     4681 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     4741 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4801 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4861 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4921 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4981 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5041 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
     5101 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5161 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5221 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5281 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5341 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5401 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5461 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5521 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5581 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5641 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5701 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5761 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5821 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5881 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     5941 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6001 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6061 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6121 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6181 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6241 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6301 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6361 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6421 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6481 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6541 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6601 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
//
