LOCUS       V034202                 7851 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V034202
VERSION     V034202
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        235..614
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        615..818
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     promoter        863..881
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     regulatory      913..922
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     CDS             922..987
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     CDS             991..2661
                     /label="HNF1B(NM_000458)"
                     /note="HNF1B(NM_000458)"
                     /gene="HNF1B"
     CDS             2701..3420
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /transl_table=1
                     /translation="M,V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL
                     TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK
                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG
                     IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV
                     LLEFVTAAGITLGMDELYK*"
                     /label="EGFP"
     polyA_signal    3451..3675
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      3721..4149
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        4163..4492
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             4559..5353
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin®)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /transl_table=1
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
                     /label="NeoR/KanR"
     polyA_signal    5527..5648
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(5697..5713)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    5721..5737
                     /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(5745..5775)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5790..5811
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(6099..6684)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6855..7715)
                     /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(7716..7820)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagccaccat ggactacaaa gaccatgacg gtgattataa agatcatgac
      961 atcgactaca aggatgacga tgacaagctt atggtgtcca agctcacgtc gctccagcaa
     1021 gaactcctga gcgccctgct gagctccggg gtcaccaagg aggtgctggt tcaggccttg
     1081 gaggagttgc tgccatcccc gaacttcggg gtgaagctgg agacgctgcc cctgtcccct
     1141 ggcagcgggg ccgagcccga caccaagccg gtcttccata ctctcaccaa cggccacgcc
     1201 aagggccgct tgtccggcga cgagggctcc gaggacggcg acgactatga cacacctccc
     1261 atcctcaagg agctgcaggc gctcaacacc gaggaggcgg cggagcagcg ggcggaggtg
     1321 gaccggatgc tcagtgagga cccttggagg gctgctaaaa tgatcaaggg ttacatgcag
     1381 caacacaaca tcccccagag ggaggtggtc gatgtcaccg gcctgaacca gtcgcacctc
     1441 tcccagcatc tcaacaaggg cacccctatg aagacccaga agcgtgccgc tctgtacacc
     1501 tggtacgtca gaaagcaacg agagatcctc cgacaattca accagacagt ccagagttct
     1561 ggaaatatga cagacaaaag cagtcaggat cagctgctgt ttctctttcc agagttcagt
     1621 caacagagcc atgggcctgg gcagtccgat gatgcctgct ctgagcccac caacaagaag
     1681 atgcgccgca accggttcaa atgggggccc gcgtcccagc aaatcttgta ccaggcctac
     1741 gatcggcaaa agaaccccag caaggaagag agagaggcct tagtggagga atgcaacagg
     1801 gcagaatgtt tgcagcgagg ggtgtccccc tccaaagccc acggcctggg ctccaacttg
     1861 gtcactgagg tccgtgtcta caactggttt gcaaaccgca ggaaggagga ggcattccgg
     1921 caaaagctgg ccatggacgc ctatagctcc aaccagactc acagcctgaa ccctctgctc
     1981 tcccacggct ccccccacca ccagcccagc tcctctcctc caaacaagct gtcaggagtg
     2041 cgctacagcc agcagggaaa caatgagatc acttcctcct caacaatcag tcaccatggc
     2101 aacagcgcca tggtgaccag ccagtcggtt ttacagcaag tctccccagc cagcctggac
     2161 ccaggccaca atctcctctc acctgatggt aaaatgatct cagtctcagg aggaggtttg
     2221 cccccagtca gcaccttgac gaatatccac agcctctccc accataatcc ccagcaatct
     2281 caaaacctca tcatgacacc cctctctgga gtcatggcaa ttgcacaaag cctcaacacc
     2341 tcccaagcac agagtgtccc tgtcatcaac agtgtggccg gcagcctggc agccctgcag
     2401 cccgtccagt tctcccagca gctgcacagc cctcaccagc agcccctcat gcagcagagc
     2461 ccaggcagcc acatggccca gcagcccttc atggcagctg tgactcagct gcagaactca
     2521 cacatgtacg cacacaagca ggaacccccc cagtattccc acacctcccg gtttccatct
     2581 gcaatggtgg tcacagatac cagcagcatc agtacactca ccaacatgtc ttcaagtaaa
     2641 cagtgtcctc tacaagcctg gtgctcgagt ggcggtggag gaagcggcgg aggtggatct
     2701 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2761 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2821 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2881 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2941 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     3001 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     3061 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     3121 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     3181 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     3241 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     3301 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     3361 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtag
     3421 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     3481 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3541 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3601 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3661 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     3721 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3781 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3841 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3901 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3961 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4021 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4081 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4141 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     4201 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     4261 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     4321 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4381 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     4441 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     4501 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     4561 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     4621 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     4681 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4741 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     4801 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     4861 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     4921 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4981 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     5041 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     5101 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     5161 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     5221 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     5281 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     5341 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     5401 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5461 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5521 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5581 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5641 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5701 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5761 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5821 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5881 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5941 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6001 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6061 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6121 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6181 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6241 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6301 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6361 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6421 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6481 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6541 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6601 tcttgatccg gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt
     6661 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6721 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6781 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6841 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6901 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6961 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7021 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7081 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7141 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7201 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7261 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7321 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7381 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7441 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7501 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7561 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7621 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7681 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7741 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7801 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
