LOCUS       V033622                10449 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V033622
VERSION     V033622
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..5129
                     /label="Nfat5(NM_133957)"
                     /note="Nfat5(NM_133957)"
                     /gene="Nfat5"
     CDS             5136..5201
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    5225..5813
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    5879..5927
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      6129..6557
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        6571..6900
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             6967..7761
                     /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    7937..8058
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(8107..8123)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    8131..8147
                     /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(8155..8185)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    8200..8221
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(8509..9097)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(9268..10128)
                     /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(10129..10233)
                     /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 ccaccatggg cggtgcttgc agctccttta
      781 ccacctcttc cagccctacc atttattcta cctcagtcac cgacagcaag gctatgcaag
      841 tggagagctg ctcctcagcc gtgggggtaa gtaacagagg ggtaagtgaa aagcagttaa
      901 ccggtaacac agttcagcag catccatcaa ccccgaagag gcacacagtt ttgtacatct
      961 caccaccacc tgaggacttg ctggataaca gtcggatgtc ctgccaggat gaggggtgtg
     1021 gattggaatc tgagcagagc tgcagtatgt ggatggagga ttccccctcc aacttcagta
     1081 acatgagcac cagttcctac aatgataaca ctgaggtacc tcgtaaatca cgaaaacgaa
     1141 atccaaagca gaggccgggg gtcaaacgac gagattgtga agaatctaat atggatatat
     1201 ttgatgccga cagtgccaaa gcacctcact atgtgctttc tcagcttacc acggacaaca
     1261 aaggcaactc aaaagctgga aatggaacat tggacagcca aaagggaact ggagtaaaga
     1321 agagccctat gttgtgcgga cagtatccgg ttaaaagtga ggggaaggag ctgaagatag
     1381 tggtacagcc tgaaacccaa caccgagccc ggtacctgac agagggcagc cgaggctctg
     1441 taaaagacag aacacagcaa ggctttccta cggtgaagct ggaaggccat aatgaaccag
     1501 tggtgttgca ggtatttgtg ggcaatgatt ctggtcgagt aaaaccacat ggattctatc
     1561 aggcctgtag agtaactggg cgaaatacaa ctccctgcaa agaagtggac attgaaggta
     1621 ccactgttat agaagttggt cttgatccta gcaacaacat gacactggcg gtggactgtg
     1681 ttggaatatt gaagctgagg aatgccgatg ttgaagccag aattggaatt gctggatcta
     1741 agaaaaagag cactcgtgcc agattggttt ttcgagttaa tatcacaagg aaagatggct
     1801 ctactctgac attgcagaca ccttcttccc ccattttatg cactcagcca gcaggagttc
     1861 ctgagatctt aaagaaaagc ttgcatagct gttcagtgaa aggagaggaa gaagtatttt
     1921 taattgggaa aaactttctg aaaggaacta aagttatttt ccaggaaaat gtttctgatg
     1981 aaaactcttg gaaatcagaa gctgaaattg acatggagtt attccatcag aaccatctta
     2041 ttgtgaaggt tcctccgtat catgaccaac atataacttt gcctgtatca gtgggaatat
     2101 atgttgtgac caatgctgga agatctcatg atgttcagcc atttacgtac actccggatc
     2161 cagcagctgg tgctttgaat gtaaatgtga aaaaggagat atctagtcca gcaagacctt
     2221 gctcttttga agaagccatg aaagcaatga aaacgactgg atgtaacgta gataaggtga
     2281 ctatccttcc taatgccctg atcactccac tcatatcaag cagtatgatt aagactgaag
     2341 atgttactcc aatggaagta acttcagaaa aaagatcttc cccaattttt cagactacaa
     2401 agagcattgg atcaacccag caaaccttag aaactatttc taacatagcg gggggtgcac
     2461 ccttctcctc accgtcgtca tcttcccact taactcctga aagtgagaat cagcagcagc
     2521 ttcagcccaa ggcatacaac ccagagaccc tgacaactat ccagacacag gacatctcac
     2581 agcccgggac ctttcccgca gtttctgctg ctagtcagct gcccagcagc gatgcactac
     2641 tgcagcaggc aacacagttt cagacaagag aagctcagtc cagagacaca atacagtcag
     2701 atacagtggt taacttgtca cagttaactg aagcatcaca gcagcagcaa tccccactac
     2761 aagaacaagc acaaacgtta cagcagcaga taccatcaaa tatttttcca tcgccaagca
     2821 gtgtcagcca gctccagagt actattcagc agctgcaagc aggaagtttc acaggcagcg
     2881 ctgctggcgg caggagtgga agcgttgatt tggtccagca ggttttagag gcacaacagc
     2941 agttatcgtc tgttctgttt tctactccag atgggaatga gaatgttcaa gaacagctta
     3001 atgcagacat tttccaagtc agtcaaattc aaaatagtgt aagccctgga atgttttctt
     3061 cagcggagtc tgcagttcac actagaccag ataacttact acctgggagg gctgacagcg
     3121 tccatcaaca gactgaaaat acactgtcta atcagcagca gcagcaacag cagcagcagc
     3181 aagtgatgga gtcatcagct gcaatggtga tggagatgca gcagagcatt tgccaagcag
     3241 ctgcccagat ccagtcagag ctgtttcctt cagctgcttc agcaagtgga agccttcagc
     3301 agtctccagt ttaccagcag ccttctcaca tgatgagtgc actgcctacc aacgaggaca
     3361 tgcagatgca gtgtgagctg ttctcgtctc cccctgcagc ttctggaaac gaaacttcca
     3421 caaccaccac gccacaggtg gcaacccctg gctccaccat gtttcagaca ccaagttcag
     3481 gagatggaga agaaactgga gcacaagcaa aacagattca gaacagcgtc tttcagacaa
     3541 tggtccaaat gcagcgcagt ggtgacagcc aacctcaagt aaacctcttt tcatctacaa
     3601 aaaatataat gagtgttcag aataatggta cccagcaaca agggaatagc ttgttccagc
     3661 aagggagcga gatgatgtca cttcagtctg ggaacttttt gcagcagtcc tctcattcac
     3721 aggctcagct ctttcatcct caaaatccta ttgctgatgc tcagaacctt tcccaggaaa
     3781 ctcaaggttc aatttttcat agtccaaatc ctattgtcca cagtcaaact tccacagcat
     3841 cctctgaaca actgcagcct tcaatgtttc actctcagaa taccattgct gtgttacagg
     3901 gctcttcagt tcctcaagac cagcagtcac ccaacatatt tctttcccaa agttctatca
     3961 ataatcttca aactaacaca gtagcccaag aagagcagat ttcatttttt gcagcacaga
     4021 actcaatttc tccacttcag tcaacatcaa acactgagca gcaagctgct ttccaacagc
     4081 agcctccaat ctcacacatc cagaccccta ttctttccca ggaacaggca caaccctctc
     4141 agcaagggtt atttcagcct caggtggccc ttggctccct gcctcctaat ccaatgcctc
     4201 aaaaccagca aggtccaatt ttccaaacac agcgcccaat agttggcatg cagagtaact
     4261 ctccatccca ggagcagcag cagcagcaac agcagcaaca gcagcagcag caacagcaac
     4321 aacaacagca acagagcatt ttattcagta atcagaatgc catggctaca atggcctccc
     4381 agaagcagcc accaccaaac atgatgttta gcccaaacca gaacccaatg gctagtcagg
     4441 agcagcagaa ccagtcaatt tttcaccagc aaagtaatat ggccccaatg aaccaagagc
     4501 agcagcccat gcaatttcag aatcagccta cggtttcctc acttcagaac ccaggtccta
     4561 cgcagtctga gtcaccacag acctccttgt tccatagctc tcctcagatc cagttggttc
     4621 aagggtcacc tagttctcaa gatcagcaag taacactctt cctctctcca gcatccatgt
     4681 ctgcattgca aaccagtata aaccaaccag acatgcagca gtctcctctt tattccccgc
     4741 agaacaacat ccctggaatc caaggaagca cttcttcacc ccagccacag gctactttat
     4801 ttcacaacac tacaggaggc acaataaacc aaatacaaaa ttctcctggc tcatctcagc
     4861 agacttcagg aatgttctta tttggcattc aaaataattg tagtcagctt ttaacctccg
     4921 gaccagctac attgccagat cagctgatgg ccataaatca gcagggccaa ccacaaaatg
     4981 agggccaatc ttctgtgaca acacttcttt ctcagcaaat gccagagact tccccactgg
     5041 cctcctctgt gaacagcagt cagaacatgg aaaagattga tttgcttgtt tcattgcaaa
     5101 gccaagggaa caatttaacc ggctcctttg gatccgacta caaagaccat gacggtgatt
     5161 ataaagatca tgacatcgac tacaaggatg acgatgacaa gtagtgaggg cccgatatct
     5221 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     5281 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     5341 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     5401 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     5461 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     5521 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     5581 gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct
     5641 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     5701 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     5761 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggaaa
     5821 cgggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     5881 gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt
     5941 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     6001 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     6061 agccaacgtc ggggcggcag gccctgccat agcagatctg cgcagctggg gctctagggg
     6121 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     6181 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     6241 tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt
     6301 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     6361 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     6421 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     6481 tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca
     6541 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     6601 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     6661 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     6721 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     6781 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     6841 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     6901 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     6961 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     7021 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     7081 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     7141 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     7201 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     7261 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     7321 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     7381 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     7441 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     7501 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     7561 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     7621 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     7681 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     7741 cttcttgacg agttcttctg agcgggactc tggggttcgc gaaatgaccg accaagcgac
     7801 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     7861 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     7921 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     7981 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     8041 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     8101 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     8161 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     8221 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     8281 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     8341 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     8401 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     8461 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     8521 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     8581 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     8641 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     8701 tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     8761 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     8821 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     8881 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     8941 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     9001 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     9061 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     9121 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     9181 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     9241 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     9301 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     9361 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     9421 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     9481 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     9541 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     9601 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     9661 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     9721 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     9781 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     9841 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     9901 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     9961 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
    10021 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
    10081 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
    10141 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
    10201 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
    10261 cgtcgacgga tcgggagatc tcccgatccc ctatggtcga ctctcagtac aatctgctct
    10321 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
    10381 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
    10441 ctgcttagg
//
