LOCUS       V032903                10377 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V032903
VERSION     V032903
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..5057
                     /label="NOS1(NM_000620)"
                     /note="NOS1(NM_000620)"
                     /gene="NOS1"
     CDS             5064..5129
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     misc_feature    5153..5741
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     polyA_signal    5807..5855
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal&nbsp;(Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     rep_origin      6057..6485
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        6499..6828
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             6895..7689
                     /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    7865..7986
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(8035..8051)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    8059..8075
                     /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(8083..8113)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    8128..8149
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(8437..9025)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(9196..10056)
                     /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(10057..10161)
                     /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 ccaccatgga ggatcacatg ttcggtgttc
      781 agcaaatcca gcccaatgtc atttctgttc gtctcttcaa gcgcaaagtt gggggcctgg
      841 gatttctggt gaaggagcgg gtcagtaagc cgcccgtgat catctctgac ctgattcgtg
      901 ggggcgccgc agagcagagt ggcctcatcc aggccggaga catcattctt gcggtcaacg
      961 gccggccctt ggtggacctg agctatgaca gcgccctgga ggtactcaga ggcattgcct
     1021 ctgagaccca cgtggtcctc attctgaggg gccctgaagg tttcaccacg cacctggaga
     1081 ccacctttac aggtgatggg acccccaaga ccatccgggt gacacagccc ctgggtcccc
     1141 ccaccaaagc cgtggatctg tcccaccagc caccggccgg caaagaacag cccctggcag
     1201 tggatggggc ctcgggtccc gggaatgggc ctcagcatgc ctacgatgat gggcaggagg
     1261 ctggctcact cccccatgcc aacggcctgg cccccaggcc cccaggccag gaccccgcga
     1321 agaaagcaac cagagtcagc ctccaaggca gaggggagaa caatgaactg ctcaaggaga
     1381 tagagcctgt gctgagcctt ctcaccagtg ggagcagagg ggtcaaggga ggggcacctg
     1441 ccaaggcaga gatgaaagat atgggaatcc aggtggacag agatttggac ggcaagtcac
     1501 acaaacctct gcccctcggc gtggagaacg accgagtctt caatgaccta tgggggaagg
     1561 gcaatgtgcc tgtcgtcctc aacaacccat attcagagaa ggagcagccc cccacctcag
     1621 gaaaacagtc ccccacaaag aatggcagcc cctccaagtg tccacgcttc ctcaaggtca
     1681 agaactggga gactgaggtg gttctcactg acaccctcca ccttaagagc acattggaaa
     1741 cgggatgcac tgagtacatc tgcatgggct ccatcatgca tccttctcag catgcaagga
     1801 ggcctgaaga cgtccgcaca aaaggacagc tcttccctct cgccaaagag tttattgatc
     1861 aatactattc atcaattaaa agatttggct ccaaagccca catggaaagg ctggaagagg
     1921 tgaacaaaga gatcgacacc actagcactt accagctcaa ggacacagag ctcatctatg
     1981 gggccaagca cgcctggcgg aatgcctcgc gctgtgtggg caggatccag tggtccaagc
     2041 tgcaggtatt cgatgcccgt gactgcacca cggcccacgg gatgttcaac tacatctgta
     2101 accatgtcaa gtatgccacc aacaaaggga acctcaggtc tgccatcacc atattccccc
     2161 agaggacaga cggcaagcac gacttccgag tctggaactc ccagctcatc cgctacgctg
     2221 gctacaagca gcctgacggc tccaccctgg gggacccagc caatgtgcag ttcacagaga
     2281 tatgcataca gcagggctgg aaaccgccta gaggccgctt cgatgtcctg ccgctcctgc
     2341 ttcaggccaa cggcaatgac cctgagctct tccagattcc tccagagctg gtgttggaag
     2401 ttcccatcag gcaccccaag tttgagtggt tcaaggacct ggggctgaag tggtacggcc
     2461 tccccgccgt gtccaacatg ctcctagaga ttggcggcct ggagttcagc gcctgtccct
     2521 tcagtggctg gtacatgggc acagagattg gtgtccgcga ctactgtgac aactcccgct
     2581 acaatatcct ggaggaagtg gccaagaaga tgaacttaga catgaggaag acgtcctccc
     2641 tgtggaagga ccaggcgctg gtggagatca atatcgcggt tctctatagc ttccagagtg
     2701 acaaagtgac cattgttgac catcactccg ccaccgagtc cttcattaag cacatggaga
     2761 atgagtaccg ctgccggggg ggctgccctg ccgactgggt gtggatcgtg ccccccatgt
     2821 ccggaagcat cacccctgtg ttccaccagg agatgctcaa ctaccggctc accccctcct
     2881 tcgaatacca gcctgatccc tggaacacgc atgtctggaa aggcaccaac gggaccccca
     2941 caaagcggcg agccattggc ttcaagaagc tagcagaagc tgtcaagttc tcggccaagc
     3001 tgatggggca ggctatggcc aagagggtga aagcgaccat cctctatgcc acagagacag
     3061 gcaaatcgca agcttatgcc aagaccttgt gtgagatctt caaacacgcc tttgatgcca
     3121 aggtgatgtc catggaagaa tatgacattg tgcacctgga acatgaaact ctggtccttg
     3181 tggtcaccag cacctttggc aatggagatc cccctgagaa tggggagaaa ttcggctgtg
     3241 ctttgatgga aatgaggcac cccaactctg tgcaggaaga aaggaagagc tacaaggtcc
     3301 gattcaacag cgtctcctcc tactctgact cccaaaaatc atcaggcgat gggcccgacc
     3361 tcagagacaa ctttgagagt gctggacccc tggccaatgt gaggttctca gtttttggcc
     3421 tcggctcacg agcataccct cacttttgcg ccttcggaca cgctgtggac accctcctgg
     3481 aagaactggg aggggagagg atcctgaaga tgagggaagg ggatgagctc tgtgggcagg
     3541 aagaggcttt caggacctgg gccaagaagg tcttcaaggc agcctgtgat gtcttctgtg
     3601 tgggagatga tgtcaacatt gaaaaggcca acaattccct catcagcaat gatcgcagct
     3661 ggaagagaaa caagttccgc ctcacctttg tggccgaagc tccagaactc acacaaggtc
     3721 tatccaatgt ccacaaaaag cgagtctcag ctgcccggct ccttagccgt caaaacctcc
     3781 agagccctaa atccagtcgg tcaactatct tcgtgcgtct ccacaccaac gggagccagg
     3841 agctgcagta ccagcctggg gaccacctgg gtgtcttccc tggcaaccac gaggacctcg
     3901 tgaatgccct gatcgagcgg ctggaggacg cgccgcctgt caaccagatg gtgaaagtgg
     3961 aactgctgga ggagcggaac acggctttag gtgtcatcag taactggaca gacgagctcc
     4021 gcctcccgcc ctgcaccatc ttccaggcct tcaagtacta cctggacatc accacgccac
     4081 caacgcctct gcagctgcag cagtttgcct ccctagctac cagcgagaag gagaagcagc
     4141 gtctgctggt cctcagcaag ggtttgcagg agtacgagga atggaaatgg ggcaagaacc
     4201 ccaccatcgt ggaggtgctg gaggagttcc catctatcca gatgccggcc accctgctcc
     4261 tgacccagct gtccctgctg cagccccgct actattccat cagctcctcc ccagacatgt
     4321 accctgatga agtgcacctc actgtggcca tcgtttccta ccgcactcga gatggagaag
     4381 gaccaattca ccacggcgta tgctcctcct ggctcaaccg gatacaggct gacgaactgg
     4441 tcccctgttt cgtgagagga gcacccagct tccacctgcc ccggaacccc caagtcccct
     4501 gcatcctcgt tggaccaggc accggcattg cccctttccg aagcttctgg caacagcggc
     4561 aatttgatat ccaacacaaa ggaatgaacc cctgccccat ggtcctggtc ttcgggtgcc
     4621 ggcaatccaa gatagatcat atctacaggg aagagaccct gcaggccaag aacaaggggg
     4681 tcttcagaga gctgtacacg gcttactccc gggagccaga caaaccaaag aagtacgtgc
     4741 aggacatcct gcaggagcag ctggcggagt ctgtgtaccg agccctgaag gagcaagggg
     4801 gccacatata cgtctgtggg gacgtcacca tggctgctga tgtcctcaaa gccatccagc
     4861 gcatcatgac ccagcagggg aagctctcgg cagaggacgc cggcgtattc atcagccgga
     4921 tgagggatga caaccgatac catgaggata tttttggagt caccctgcga acgtacgaag
     4981 tgaccaaccg ccttagatct gagtccattg ccttcattga agagagcaaa aaagacaccg
     5041 atgaggtttt cagctccgga tccgactaca aagaccatga cggtgattat aaagatcatg
     5101 acatcgacta caaggatgac gatgacaagt agtgagggcc cgatatctcg acaatcaacc
     5161 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     5221 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     5281 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     5341 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     5401 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     5461 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     5521 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     5581 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     5641 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     5701 ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggaaacg ggggaggcta
     5761 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     5821 cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     5881 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     5941 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     6001 ggcggcaggc cctgccatag cagatctgcg cagctggggc tctagggggt atccccacgc
     6061 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     6121 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     6181 cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc
     6241 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     6301 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     6361 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     6421 gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     6481 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     6541 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     6601 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     6661 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     6721 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     6781 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     6841 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     6901 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     6961 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     7021 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     7081 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     7141 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     7201 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     7261 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     7321 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     7381 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     7441 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     7501 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     7561 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     7621 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     7681 ttcttctgag cgggactctg gggttcgcga aatgaccgac caagcgacgc ccaacctgcc
     7741 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     7801 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     7861 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     7921 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     7981 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     8041 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     8101 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     8161 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     8221 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     8281 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     8341 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     8401 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     8461 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     8521 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     8581 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     8641 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     8701 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     8761 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     8821 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     8881 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     8941 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     9001 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     9061 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     9121 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     9181 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     9241 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     9301 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     9361 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     9421 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     9481 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     9541 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     9601 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     9661 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     9721 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     9781 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     9841 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     9901 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     9961 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
    10021 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
    10081 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
    10141 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
    10201 gggagatctc ccgatcccct atggtcgact ctcagtacaa tctgctctga tgccgcatag
    10261 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
    10321 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagg
//
