LOCUS       V024983                 7616 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V024983
VERSION     V024983
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..3825
                     /label="GRK3(NM_005160)"
                     /note="GRK3(NM_005160)"
                     /gene="GRK3"
     CDS             3832..3852
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /transl_table=1
                     /translation="ENLYFQS"
                     /label="TEV site"
     CDS             3853..4566
                     /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             4567..4590
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin®, an engineered
                     form of streptavidin"
                     /transl_table=1
                     /translation="WSHPQFEK"
                     /label="Strep-Tag II"
     CDS             4609..4632
                     /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    4704..4759
                     /note="rabbit&nbsp;β-globin polyadenylation signal (Gil and
                     Proudfoot, 1987)"
                     /label="β-globin poly(A) signal"
     primer_bind     complement(5119..5135)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    5143..5159
                     /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(5167..5197)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5212..5233
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     promoter        5292..5487
                     /note="SV40 early promoter"
                     /label="SV40 promoter"
     polyA_signal    5493..5627
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      complement(5866..6454)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6625..7485)
                     /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(7486..7590)
                     /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 gatggcggac ctggaggctg tgctggccga tgtcagttac
     1801 ctgatggcca tggagaagag caaggcgacc ccggccgccc gcgccagcaa gaggatcgtc
     1861 ctgccggagc ccagtatccg gagtgtgatg cagaagtacc ttgcagagag aaatgaaata
     1921 acctttgaca agattttcaa tcagaaaatt ggtttcttgc tatttaaaga tttttgtttg
     1981 aatgaaatta atgaagctgt acctcaggtg aagttttatg aagagataaa ggaatatgaa
     2041 aaacttgata atgaggaaga ccgcctttgc agaagtcgac aaatttatga tgcctacatc
     2101 atgaaggaac ttctttcctg ttcacatcct ttctcaaagc aagctgtaga acacgtacaa
     2161 agtcatttat ccaagaaaca agtgacatca actctttttc agccatacat agaagaaatt
     2221 tgtgaaagcc ttcgaggtga catttttcaa aaatttatgg aaagtgacaa gttcactaga
     2281 ttttgtcagt ggaaaaacgt tgaattaaat atccatttga ccatgaatga gttcagtgtg
     2341 cataggatta ttggacgagg aggattcggg gaagtttatg gttgcaggaa agcagacact
     2401 ggaaaaatgt atgcaatgaa atgcttagat aagaagagga tcaaaatgaa acaaggagaa
     2461 acattagcct taaatgaaag aatcatgttg tctcttgtca gcacaggaga ctgtcctttc
     2521 attgtatgta tgacctatgc cttccatacc ccagataaac tctgcttcat cctggatctg
     2581 atgaacgggg gcgatttgca ctaccacctt tcacaacacg gtgtgttctc tgagaaggag
     2641 atgcggtttt atgccactga aatcattctg ggtctggaac acatgcacaa tcggtttgtt
     2701 gtctacagag atttgaagcc agcaaatatt ctcttggatg aacatggaca cgcaagaata
     2761 tcagatcttg gtcttgcctg cgatttttcc aaaaagaagc ctcatgcgag tgttggcacc
     2821 catgggtaca tggctcccga ggtgctgcag aaggggacgg cctatgacag cagtgccgac
     2881 tggttctccc tgggctgcat gcttttcaaa cttctgagag gtcacagccc tttcagacaa
     2941 cataaaacca aagacaagca tgaaattgac cgaatgacac tcaccgtgaa tgtggaactt
     3001 ccagacacct tctctcctga actgaagtcc cttttggagg gcttgcttca gcgagacgtt
     3061 agcaagcggc tgggctgtca cggaggcggc tcacaggaag taaaagagca cagctttttc
     3121 aaaggtgttg actggcagca tgtctactta caaaagtacc caccaccctt gattcctccc
     3181 cggggagaag tcaatgctgc tgatgccttt gatattggct catttgatga agaggatacc
     3241 aaagggatta agctacttga ttgcgaccaa gaactctaca agaacttccc tttggtcatc
     3301 tctgaacgct ggcagcaaga agtaacggaa acagtttatg aagcagtaaa tgcagacaca
     3361 gataaaatcg aggccaggaa gagagctaaa aataagcaac ttggccacga agaagattac
     3421 gctctgggga aggactgtat tatgcacggg tacatgctga aactgggaaa cccatttctg
     3481 actcagtggc agcgtcgcta tttttacctc tttccaaata gacttgaatg gagaggagag
     3541 ggagagtccc ggcaaaattt actgacaatg gaacagattc tctctgtgga agaaactcaa
     3601 attaaagaca aaaaatgcat tttgttcaga ataaaaggag ggaaacaatt tgtcttgcaa
     3661 tgtgagagtg atccagagtt tgtgcagtgg aagaaagagt tgaacgaaac cttcaaggag
     3721 gcccagcggc tattgcgtcg tgccccgaag ttcctcaaca aacctcggtc aggtactgtg
     3781 gagctcccaa agccatccct ctgtcacaga aacagcaacg gcctcgaatt cgaaaacctg
     3841 tacttccaat ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     3901 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     3961 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     4021 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     4081 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     4141 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     4201 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     4261 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     4321 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     4381 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     4441 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     4501 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     4561 tacaagtgga gccacccgca gttcgagaag ggcggcggca gcagcgcctg gagccacccg
     4621 cagttcgaga agtgaagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc
     4681 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg
     4741 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca
     4801 gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa
     4861 aggtggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc
     4921 catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt
     4981 tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc
     5041 tgactactcc cagtcatagc tgtccctctt ctcttatgaa gatccctcga cctgcagccc
     5101 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5161 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5221 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5281 ccagcggatc cgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     5341 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     5401 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     5461 ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt
     5521 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5581 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg
     5641 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5701 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5761 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5821 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5881 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5941 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6001 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6061 atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6121 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6181 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6241 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6301 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6361 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6421 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6481 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6541 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6601 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6661 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6721 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6781 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6841 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6901 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6961 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7021 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7081 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7141 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7201 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7261 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7321 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7381 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7441 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7501 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7561 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctg
//
