LOCUS       V035099                 6859 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V035099
VERSION     V035099
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"
     CDS             964..2349
                     /label="SHH(NM_000193)"
                     /note="SHH(NM_000193)"
                     /gene="SHH"
     CDS             2356..2421
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     polyA_signal    2459..2683
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      2729..3157
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        3171..3500
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             3567..4361
                     /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    4535..4656
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(4705..4721)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    4729..4745
                     /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(4753..4783)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    4798..4819
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(5107..5692)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(5863..6723)
                     /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(6724..6828)
                     /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 aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcgcc
      961 accatgctgc tgctggcgag atgtctgctg ctagtcctcg tctcctcgct gctggtatgc
     1021 tcgggactgg cgtgcggacc gggcaggggg ttcgggaaga ggaggcaccc caaaaagctg
     1081 acccctttag cctacaagca gtttatcccc aatgtggccg agaagaccct aggcgccagc
     1141 ggaaggtatg aagggaagat ctccagaaac tccgagcgat ttaaggaact cacccccaat
     1201 tacaaccccg acatcatatt taaggatgaa gaaaacaccg gagcggacag gctgatgact
     1261 cagaggtgta aggacaagtt gaacgctttg gccatctcgg tgatgaacca gtggccagga
     1321 gtgaaactgc gggtgaccga gggctgggac gaagatggcc accactcaga ggagtctctg
     1381 cactacgagg gccgcgcagt ggacatcacc acgtctgacc gcgaccgcag caagtacggc
     1441 atgctggccc gcctggcggt ggaggccggc ttcgactggg tgtactacga gtccaaggca
     1501 catatccact gctcggtgaa agcagagaac tcggtggcgg ccaaatcggg aggctgcttc
     1561 ccgggctcgg ccacggtgca cctggagcag ggcggcacca agctggtgaa ggacctgagc
     1621 cccggggacc gcgtgctggc ggcggacgac cagggccggc tgctctacag cgacttcctc
     1681 actttcctgg accgcgacga cggcgccaag aaggtcttct acgtgatcga gacgcgggag
     1741 ccgcgcgagc gcctgctgct caccgccgcg cacctgctct ttgtggcgcc gcacaacgac
     1801 tcggccaccg gggagcccga ggcgtcctcg ggctcggggc cgccttccgg gggcgcactg
     1861 gggcctcggg cgctgttcgc cagccgcgtg cgcccgggcc agcgcgtgta cgtggtggcc
     1921 gagcgtgacg gggaccgccg gctcctgccc gccgctgtgc acagcgtgac cctaagcgag
     1981 gaggccgcgg gcgcctacgc gccgctcacg gcccagggca ccattctcat caaccgggtg
     2041 ctggcctcgt gctacgcggt catcgaggag cacagctggg cgcaccgggc cttcgcgccc
     2101 ttccgcctgg cgcacgcgct cctggctgca ctggcgcccg cgcgcacgga ccgcggcggg
     2161 gacagcggcg gcggggaccg cgggggcggc ggcggcagag tagccctaac cgctccaggt
     2221 gctgccgacg ctccgggtgc gggggccacc gcgggcatcc actggtactc gcagctgctc
     2281 taccaaatag gcacctggct cctggacagc gaggccctgc acccgctggg catggcggtc
     2341 aagtccagcc tcgaggacta caaagaccat gacggtgatt ataaagatca tgacatcgac
     2401 tacaaggatg acgatgacaa gtagtgaggg cccgtttaaa cccgctgatc agcctcgact
     2461 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2521 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2581 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2641 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2701 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     2761 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2821 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2881 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2941 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3001 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3061 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     3121 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     3181 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3241 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3301 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     3361 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3421 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3481 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     3541 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3601 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3661 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3721 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     3781 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3841 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3901 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3961 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4021 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4081 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4141 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4201 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4261 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4321 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     4381 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4441 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4501 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4561 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4621 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4681 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4741 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4801 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4861 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4921 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4981 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5041 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5101 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5161 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5221 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5281 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5341 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5401 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5461 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5521 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5581 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5641 cggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5701 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5761 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5821 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5881 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5941 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6001 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6061 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6121 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6181 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6241 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6301 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6361 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6421 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6481 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6541 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6601 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6661 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6721 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6781 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6841 aaaagtgcca cctgacgtc
//
