LOCUS       V035679                 7528 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V035679
VERSION     V035679
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..3048
                     /label="LIMK2(NM_001031801)"
                     /note="LIMK2(NM_001031801)"
                     /gene="LIMK2"
     polyA_signal    3128..3352
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      3398..3826
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        3840..4169
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             4236..5030
                     /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    5204..5325
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(5374..5390)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    5398..5414
                     /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(5422..5452)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5467..5488
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(5776..6361)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6532..7392)
                     /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(7393..7497)
                     /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 atggggagtt acttgtcagt cccggcttac
     1021 ttcacctcca gagacctgtt tcggtgttca gaatgccagg attccctcac caactggtac
     1081 tatgagaagg atgggaagct ctactgcccc aaggactact gggggaagtt tggggagttc
     1141 tgtcatgggt gctccctgct gatgacaggg ccttttatgg tggctgggga gttcaagtac
     1201 cacccagagt gctttgcctg tatgagctgc aaggtgatca ttgaggatgg ggatgcatat
     1261 gcactggtgc agcatgccac cctctactgt gggaagtgcc acaatgaggt ggtgctggca
     1321 cccatgtttg agagactctc cacagagtct gttcaggagc agctgcccta ctctgtcacg
     1381 ctcatctcca tgccggccac cactgaaggc aggcggggct tctccgtgtc cgtggagagt
     1441 gcctgctcca actacgccac cactgtgcaa gtgaaagagg tcaaccggat gcacatcagt
     1501 cccaacaatc gaaacgccat ccaccctggg gaccgcatcc tggagatcaa tgggaccccc
     1561 gtccgcacac ttcgagtgga ggaggtggag gatgcaatta gccagacgag ccagacactt
     1621 cagctgttga ttgaacatga ccccgtctcc caacgcctgg accagctgcg gctggaggcc
     1681 cggctcgctc ctcacatgca gaatgccgga cacccccacg ccctcagcac cctggacacc
     1741 aaggagaatc tggaggggac actgaggaga cgttccctaa ggcgcagtaa cagtatctcc
     1801 aagtcccctg gccccagctc cccaaaggag cccctgctgt tcagccgtga catcagccgc
     1861 tcagaatccc ttcgttgttc cagcagctat tcacagcaga tcttccggcc ctgtgaccta
     1921 atccatgggg aggtcctggg gaagggcttc tttgggcagg ctatcaaggt gacacacaaa
     1981 gccacgggca aagtgatggt catgaaagag ttaattcgat gtgatgagga gacccagaaa
     2041 acttttctga ctgaggtgaa agtgatgcgc agcctggacc accccaatgt gctcaagttc
     2101 attggtgtgc tgtacaagga taagaagctg aacctcctga cagagtacat tgaggggggc
     2161 acactgaagg actttctgcg cagtatggat ccgttcccct ggcagcagaa ggtcaggttt
     2221 gccaaaggaa tcgcctccgg aatggcctat ttgcactcta tgtgcatcat ccaccgggat
     2281 ctgaactcgc acaactgcct catcaagttg gacaagactg tggtggtggc agactttggg
     2341 ctgtcacggc tcatagtgga agagaggaaa agggccccca tggagaaggc caccaccaag
     2401 aaacgcacct tgcgcaagaa cgaccgcaag aagcgctaca cggtggtggg aaacccctac
     2461 tggatggccc ctgagatgct gaacggaaag agctatgatg agacggtgga tatcttctcc
     2521 tttgggatcg ttctctgtga gatcattggg caggtgtatg cagatcctga ctgccttccc
     2581 cgaacactgg actttggcct caacgtgaag cttttctggg agaagtttgt tcccacagat
     2641 tgtcccccgg ccttcttccc gctggccgcc atctgctgca gactggagcc tgagagcaga
     2701 gccccccccg gggccgcagg agagggcccg ggctgcgcgg atgatgaggg cccagtgagg
     2761 cgccaaggga aggtcaccat caagtatgac cccaaggagc tacggaagca cctcaaccta
     2821 gaggagtgga tcctggagca gctcacgcgc ctctacgact gccaggaaga ggagatctca
     2881 gaactagaga ttgacgtgga tgagctcctg gacatggaga gtgacgatgc ctgggcttcc
     2941 agggtcaagg agctgctggt tgactgttac aaacccacag aggccttcat ctctggcctg
     3001 ctggacaaga tccgggccat gcagaagctg agcacacccc agaagaagtg agaattctgc
     3061 agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca
     3121 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3181 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3241 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3301 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     3361 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     3421 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3481 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3541 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3601 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3661 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3721 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3781 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     3841 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3901 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3961 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     4021 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4081 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     4141 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     4201 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     4261 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     4321 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     4381 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     4441 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     4501 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     4561 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4621 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     4681 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4741 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     4801 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4861 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4921 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4981 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     5041 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     5101 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     5161 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     5221 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     5281 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     5341 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5401 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5461 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5521 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5581 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5641 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5701 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5761 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5821 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5881 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5941 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6001 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6061 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6121 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6181 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6241 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6301 cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6361 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6421 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6481 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6541 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6601 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6661 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6721 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6781 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6841 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6901 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6961 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7021 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7081 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7141 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7201 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7261 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7321 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7381 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7441 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7501 atttccccga aaagtgccac ctgacgtc
//
