LOCUS       V024581                 6816 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V024581
VERSION     V024581
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        50..429
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        430..633
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     promoter        678..696
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     CDS             756..1496
                     /label="GZMB(NM_004131)"
                     /note="GZMB(NM_004131)"
                     /gene="GZMB"
     CDS             1503..1568
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    1592..2180
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    2246..2294
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      2496..2924
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        2938..3267
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             3334..4128
                     /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    4304..4425
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(4474..4490)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    4498..4514
                     /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(4522..4552)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    4567..4588
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(4876..5464)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(5635..6495)
                     /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(6496..6600)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      661 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta
      721 aacttaagct tggtaccgag ctcgaattcg ccaccatgca accaatcctg cttctgctgg
      781 ccttcctcct gctgcccagg gcagatgcag gggagatcat cgggggacat gaggccaagc
      841 cccactcccg cccctacatg gcttatctta tgatctggga tcagaagtct ctgaagaggt
      901 gcggtggctt cctgatacga gacgacttcg tgctgacagc tgctcactgt tggggaagct
      961 ccataaatgt caccttgggg gcccacaata tcaaagaaca ggagccgacc cagcagttta
     1021 tccctgtgaa aagacccatc ccccatccag cctataatcc taagaacttc tccaacgaca
     1081 tcatgctact gcagctggag agaaaggcca agcggaccag agctgtgcag cccctcaggc
     1141 tacctagcaa caaggcccag gtgaagccag ggcagacatg cagtgtggcc ggctgggggc
     1201 agacggcccc cctgggaaaa cactcacaca cactacaaga ggtgaagatg acagtgcagg
     1261 aagatcgaaa gtgcgaatct gacttacgcc attattacga cagtaccatt gagttgtgcg
     1321 tgggggaccc agagattaaa aagacttcct ttaaggggga ctctggaggc cctcttgtgt
     1381 gtaacaaggt ggcccagggc attgtctcct atggacgaaa caatggcatg cctccacgag
     1441 cctgcaccaa agtctcaagc tttgtacact ggataaagaa aaccatgaaa cgctacggat
     1501 ccgactacaa agaccatgac ggtgattata aagatcatga catcgactac aaggatgacg
     1561 atgacaagta gtgagggccc gatatctcga caatcaacct ctggattaca aaatttgtga
     1621 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     1681 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     1741 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     1801 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     1861 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     1921 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     1981 ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     2041 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     2101 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     2161 cctttgggcc gcctccccgc ctggaaacgg gggaggctaa ctgaaacacg gaaggagaca
     2221 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt
     2281 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     2341 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     2401 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     2461 agatctgcgc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2521 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2581 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2641 tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2701 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2761 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2821 actcaaccct atctcggtct attcttttga tttataaggg attttgggga tttcggccta
     2881 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2941 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3001 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3061 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3121 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3181 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3241 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3301 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3361 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3421 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3481 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3541 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3601 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3661 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3721 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3781 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3841 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3901 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3961 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4021 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4081 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4141 ggttcgcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4201 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4261 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     4321 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4381 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     4441 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4501 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4561 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4621 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4681 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4741 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4801 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4861 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4921 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4981 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5041 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     5101 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5161 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5221 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5281 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     5341 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5401 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5461 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5521 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5581 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5641 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5701 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5761 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5821 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5881 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5941 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6001 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6061 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6121 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6181 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6241 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6301 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6361 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6421 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6481 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6541 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6601 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     6661 tggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     6721 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     6781 ggcttgaccg acaattgcat gaagaatctg cttagg
//
