LOCUS       V032995                10087 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V032995
VERSION     V032995
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..5607
                     /label="ZFYVE16(NM_001105251)"
                     /note="ZFYVE16(NM_001105251)"
                     /gene="ZFYVE16"
     polyA_signal    5687..5911
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      5957..6385
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        6399..6728
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             6795..7589
                     /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    7763..7884
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(7933..7949)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    7957..7973
                     /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(7981..8011)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    8026..8047
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(8335..8920)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(9091..9951)
                     /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(9952..10056)
                     /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 atggacagtt attttaaagc agctgtcagt
     1021 gacttggaca aactccttga tgattttgaa cagaacccag atgaacaaga ttatctccaa
     1081 gatgtacaaa atgcatatga ttctaaccac tgctcagttt cttcagagtt ggcttcctca
     1141 cagcgaactt cattgctccc aaaagaccaa gagtgcgtta atagttgtgc ctcatcagaa
     1201 acaagctatg gaacaaatga gagttccctg aatgaaaaaa cactcaaggg acttacttct
     1261 atacaaaatg aaaaaaatgt aacaggactt gatcttcttt cttctgtgga tggtggtact
     1321 tcagatgaaa tccagccgtt atatatggga cgatgtagta aacctatctg tgatctgata
     1381 agtgacatgg gtaacttagt tcatgcaacc aatagtgaag aagatattaa aaaattattg
     1441 ccagatgatt ttaagtctaa tgcagattcc ttgattggat tggatttatc ttcagtgtca
     1501 gatactccct gtgtttcttc aacagaccat gatagtgata ctgtcagaga acaacagaat
     1561 gatatcagtt ctgaattaca aaatagagaa atcggaggaa tcaaagaatt gggtataaaa
     1621 gtagatacaa cactttcaga ttcctataat tacagtggaa cagaaaattt aaaagataaa
     1681 aagatcttta atcagttaga atcaattgtt gattttaaca tgtcatctgc tttgactcga
     1741 caaagttcca aaatgtttca tgccaaagac aagctacaac acaagagcca gccatgtgga
     1801 ttactaaaag atgttggctt agtaaaagag gaagtagatg tggcagtcat aactgccgca
     1861 gaatgtttaa aagaagaggg caagacaagt gctttgacct gcagccttcc gaaaaatgaa
     1921 gatttatgct taaatgattc aaattcaaga gatgaaaatt tcaaattacc tgacttttcc
     1981 tttcaggaag ataagactgt tataaaacaa tctgcacaag aagactcaaa aagtttagac
     2041 cttaaggata atgatgtaat ccaagattcc tcttcagctt tacatgtttc cagtaaagat
     2101 gtgccgtcct cattgtcctg tcttcctgcg tctgggtcta tgtgtggatc attaattgaa
     2161 agtaaagcac ggggtgattt tttacctcag catgaacata aagataatat acaagatgca
     2221 gtgactatac atgaagaaat acagaacagt gttgttctag gtggggaacc attcaaagag
     2281 aatgatcttt tgaaacagga aaaatgtaaa agcatactcc ttcagtcatt aattgaaggg
     2341 atggaagaca gaaagataga tcctgaccag acagtaatca gagctgagtc tttggatggt
     2401 ggtgacacca gttctacagt tgtagaatct caagaggggc tttctggcac tcatgtccca
     2461 gagtcttctg attgttgtga aggttttatt aatacttttt caagcaatga tatggatggg
     2521 caagacttag attactttaa tattgatgaa ggcgcaaaaa gtggcccact aattagtgat
     2581 gctgaacttg atgcctttct gacagaacag tatcttcaga ccactaacat aaagtctttt
     2641 gaagaaaatg taaatgactc taaatcgcaa atgaatcaga tagatatgaa aggcttagat
     2701 gatggaaaca tcaataatat atatttcaat gcagaagcag gagctattgg ggaaagtcat
     2761 ggtattaata taatttgtga aatagttgat aaacaaaata caatagaaaa tggcctttct
     2821 ttaggagaaa aaagcactat tccagttcaa caagggttac ctaccagtaa gtctgagatt
     2881 acaaatcaat tatcggtctc tgatattaac agtcaatctg ttggaggggc cagacctaag
     2941 caattgttta gccttccatc aagaacaagg agttcaaagg acctgaataa gccagatgtt
     3001 ccagatacaa tagaaagtga acccagcaca gcagataccg ttgttccaat cacttgtgct
     3061 atagattcta cagctgatcc acaggttagc ttcaactcta attacattga tatagaaagt
     3121 aattctgaag gtggatctag tttcgtaact gcaaatgaag attctgtacc tgaaaacact
     3181 tgcaaagaag gcttggtttt gggccagaaa cagcctactt gggttcctga ttcagaagct
     3241 ccaaactgta tgaactgcca agtcaaattt acttttacca aacggcgaca ccattgccga
     3301 gcatgtggga aagtattttg tggtgtctgt tgtaatagga agtgtaaact gcaatatcta
     3361 gaaaaggaag caagagtatg tgtagtctgc tatgaaacta ttagtaaagc tcaggcattt
     3421 gaaaggatga tgagtccaac tggttctaat cttaagtcta atcattctga tgaatgtact
     3481 actgtccagc ctcctcagga gaaccaaaca tccagtatac cttcaccagc aactttgcca
     3541 gtctcagcac ttaaacaacc aggtgttgaa ggactatgtt ccaaagaaca gaagagagta
     3601 tggtttgcag atggtatatt gcccaatggt gaagttgcag atacaacaaa attatcatct
     3661 ggaagtaaaa gatgttctga agactttagt cctctctcac ctgatgtgcc tatgacagta
     3721 aacacagtgg atcattccca ttctactaca gtggaaaagc caaacaatga gacaggagat
     3781 attacaagaa atgagataat tcagagtcct atttctcagg ttccatcagt ggaaaaattg
     3841 tctatgaaca caggaaatga ggggttacct acttctggtt catttacact agatgatgat
     3901 gtttttgcag aaactgaaga accatctagt cctactggtg tcttagttaa cagcaattta
     3961 cctattgcta gtatttcaga ttataggtta ctgtgtgata ttaacaagta tgtctgcaat
     4021 aagattagtc ttctacctaa tgatgaggac agtttgcccc cacttctggt tgcatctgga
     4081 gaaaagggat cagtgcctgt agtagaagaa catccatctc atgagcagat cattttgctt
     4141 cttgaaggtg aaagctttca tcctgttaca tttgtcctaa atgctaatct actcgtgaat
     4201 gtcaaattca tattttattc ctcagacaaa tattggtact tttcaaccaa tggattgcat
     4261 ggcttgggac aggcagaaat tattattcta ttgttatgtt tgccaaatga agatactatt
     4321 cctaaggaca tcttcagact atttatcacc atatataagg atgctctaaa aggaaaatac
     4381 atagaaaact tggacaatat tacctttact gagagttttc tcagtagcaa ggatcacgga
     4441 ggattcctgt ttattacacc tacttttcag aaacttgatg atctctcatt accaagtaat
     4501 ccttttcttt gtggaattct tatccagaag cttgagattc cctgggcaaa ggtttttcct
     4561 atgcgtttaa tgttgagatt gggtgcagaa tataaagcat atcctgctcc tctaacaagc
     4621 atcagaggcc gaaaacctct ttttggagaa ataggacaca ctattatgaa cttacttgtt
     4681 gaccttcgaa attaccagta taccttgcat aatatagatc aactgttgat tcatatggaa
     4741 atgggaaaaa gctgcataaa aataccacgg aaaaagtaca gtgatgtaat gaaagtacta
     4801 aattcttcca atgagcatgt cattagcatt ggagcaagtt tcagtacaga agcagattct
     4861 catctagtct gtatacagaa tgatggaatt tatgaaacac aggccaacag tgccactggc
     4921 catcctagaa aagtgacagg tgcaagtttt gtggtattca atggagctct aaaaacatct
     4981 tcaggatttc ttgctaagtc cagcatagtt gaagatggct taatggtaca aataactcca
     5041 gagaccatga atggcttgcg gctagcttta cgagaacaga aagactttaa aattacatgt
     5101 gggaaagttg atgcagtaga cctgagagaa tacgtggata tctgctgggt agatgctgaa
     5161 gaaaaaggaa acaaaggagt tatcagttca gtggatggaa tatcattaca aggatttcca
     5221 agtgaaaaaa taaaactgga agcagatttt gaaaccgatg agaagattgt aaaatgtacc
     5281 gaggtgttct actttctaaa ggaccaggat ttatctattt tatcaacttc ttatcagttt
     5341 gcaaaagaaa tagccatggc ttgtagtgct gcgctgtgcc ctcacctgaa aactctaaaa
     5401 agtaatggga tgaataaaat tggactcaga gtttccattg acactgatat ggttgaattt
     5461 caggcaggat ctgaaggcca acttctgcct cagcattatc taaatgatct tgatagtgct
     5521 ctgatacctg tgatccatgg tgggacctcc aactctagtt taccattaga aatagaatta
     5581 gtgtttttca ttatagaaca tcttttttag gaattctgca gatatccagc acagtggcgg
     5641 ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     5701 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     5761 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     5821 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     5881 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     5941 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6001 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6061 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6121 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6181 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     6241 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     6301 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     6361 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     6421 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     6481 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     6541 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     6601 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     6661 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     6721 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     6781 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     6841 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     6901 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     6961 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     7021 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     7081 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     7141 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     7201 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     7261 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     7321 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     7381 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     7441 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     7501 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     7561 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     7621 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     7681 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     7741 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     7801 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     7861 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     7921 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     7981 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     8041 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     8101 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     8161 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     8221 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     8281 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     8341 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     8401 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     8461 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     8521 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     8581 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     8641 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     8701 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     8761 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     8821 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtttttttgt
     8881 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     8941 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     9001 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     9061 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     9121 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     9181 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     9241 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     9301 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     9361 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     9421 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     9481 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     9541 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     9601 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     9661 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     9721 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     9781 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     9841 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     9901 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     9961 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
    10021 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
    10081 tgacgtc
//
