LOCUS       40924_775        9966 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector EN.CFP.SV40, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9966)
  AUTHORS   Chen SX, Osipovich AB, Ustione A, Potter LA, Hipkens S, Gangula R, 
            Yuan W, Piston DW, Magnuson MA.
  TITLE     Quantification of factors influencing fluorescent protein expression
            using RMCE to generate an allelic series in the ROSA26 locus in mice
  JOURNAL   Dis Model Mech 4 (4), 537-547 (2011)
  PUBMED    21324933
REFERENCE   2  (bases 1 to 9966)
  AUTHORS   Chen SX, Osipovich AB, Magnuson MA.
  TITLE     Direct Submission
  JOURNAL   Submitted (16-JUL-2010) Molecular Physiology and Biophysics, 
            Vanderbilt University, 9475 MRB IV, 2214 Garland Avenue, Nashville, 
            TN 37232, USA
REFERENCE   3  (bases 1 to 9966)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9966)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Dis Model 
            Mech"; date: "2011"; volume: "4"; issue: "4"; pages: "537-547"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (16-JUL-2010) Molecular Physiology and Biophysics, Vanderbilt 
            University, 9475 MRB IV, 2214 Garland Avenue, Nashville, TN 37232, 
            USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(13..31)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(52..68)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    76..92
                     /label=lac operator
                     /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-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(100..130)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(145..166)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(454..1042)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1216..2073)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(2074..2178)
                     /label=AmpR promoter
     rep_origin      complement(2253..2708)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     2850..2866
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2873..2891
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2906..2939
                     /label=lox71
                     /note="Left element (LE) mutant of loxP (Araki et al.,
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2957..7037
                     /label=Rosa 26 promoter
                     /note="Rosa 26 promoter"
                     /regulatory_class="promoter"
     5'UTR           7050..7092
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             7103..7819
                     /label=mCerulean
                     /note="enhanced monomeric variant of CFP (Rizzo et al.,
                     2004)"
     polyA_signal    7931..8012
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    8045..8078
                     /label=FRT (minimal)
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     promoter        8142..8641
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     promoter        8653..8700
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             8719..9519
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
     polyA_signal    9560..9784
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    9842..9875
                     /label=FRT site
                     /note="FRT site"
     protein_bind    9842..9875
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     protein_bind    9928..9961
                     /label=lox2272
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
ORIGIN
        1 cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct
       61 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
      121 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
      181 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      241 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
      301 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
      361 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
      421 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
      481 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
      541 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
      601 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
      661 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
      721 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
      781 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
      841 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
      901 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
      961 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1021 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1081 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1141 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     1201 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     1261 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     1321 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     1381 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     1441 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     1501 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     1561 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     1621 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     1681 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     1741 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     1801 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     1861 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     1921 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     1981 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     2041 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     2101 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2161 acaaataggg gttccgcgca catttccccg aaaagtgcca caaaaataaa caaatagggg
     2221 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     2281 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2341 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     2401 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2461 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     2521 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     2581 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2641 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     2701 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     2761 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     2821 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga
     2881 ctcactatag ggcgaattgg agctcataac ttcgtataat gtatgctata cgaacggtag
     2941 cggccgcagg gcccatcgat actgactact attgccttat gtttcaaagt tccaccttca
     3001 tcaaaagggt cagaataatt tctaaagtat acaggcttct agatactaag cagtatcaag
     3061 atgtaggcat ttttaagtgt gtcagttcaa gtaatccttc taagaaaaaa gcctcttgat
     3121 cttccagaga ttactgtaag accacaatca gatccaaaaa aacaggaaat aaaagtggta
     3181 gtcattattt tctaagggaa cttgtatatt ataattaagc tcaaaacact tgcaccttaa
     3241 ctgtaaatac cttgaactgt aatatcagta tgagtgatat catacttatg ggatacagca
     3301 cctaatctac gtaacaaaaa tattattaca accatctaaa acaatcatta acagagtttt
     3361 gttttatgga cttgctttta gcactggaaa aacaatatga actacatttg tgaattcaaa
     3421 atactctaca atgtttcttc attttttgct acaaattcta gaaccattac atttatggga
     3481 tctaaataaa tttgtacaaa attaaacaga ctcccgaagg aaaaggcatt ggttgcagta
     3541 ataatcctag ggaggaaaaa ccctacgatt ttatgtgctg cacatgcctg ggtgatggtt
     3601 cagtttcaaa tattccttgc cagacaaact acttaaaaag gcttaatcaa agatcaccct
     3661 ggttctattt ggttaggggt agggtgggct tagaatggac tcttgctgcc ttcttagtat
     3721 cttgaactaa acttactcaa tttctgaata ttccttctgt gtctctgatg gattactgcc
     3781 aaaacacatc tttgttcggt ggaaagtcta taatccaaca catacacgat taatccaatt
     3841 tataggtcag atatcagtaa gtatatctaa tctggagagg tctacagatt aatttagaat
     3901 taacaacaaa ccaagcatta atccaaaagc agctcttttt agtttaggca tccaaatata
     3961 tagtaagatg tttcaaagaa catcaatcta ttcacaaagg taaatacggc ctaatacgga
     4021 gagacaaata caaatctgaa atgactcaag caattaaaag atggaggaat tcatgtcact
     4081 tgcactggga aaacaaaaca aagatcctcc tcaccctatt acccacatta ctatttttag
     4141 gttttttagg ggttaaaaaa gatcaaaaca ctaatgaact ttaagtcctg tgaagggtaa
     4201 aacctcagat agtaacaaaa agcttccaac ccctcctcaa acaaaaaacc ccaagtcttt
     4261 aactttgatc cagttttcag atgctgatat ccataaatgg atacagttat gaattgctaa
     4321 ttctggtctc ttcactagca aaaagcaaag cagctcagca gtacaatttc ccaggaaagc
     4381 aagcaaggtt tctttccagc ctgagcagcc atcactaagt gcagttccct gcagccaaca
     4441 gcattaatgg acgctgcact gctgtccttc cctggagaca gcagccagca ctactcaagc
     4501 ttctcacgta gcaaccagag ctccagagcc agcagctgct gccgccttgt atactcactc
     4561 ctgtgatcca acacaggagc aaccttttct ttaccccacc cccacttctt aacacacttt
     4621 tttttggggg ggggggggaa caagtgctcc atgctggaag gattggaact atgcttttag
     4681 aaaggaacaa tcctaaggtc acttttaaat tgaggtcttt gatttgaaaa tcaacaaata
     4741 ccaaattcca aatattcgtt ttaattaaac cagcaatgtg gatataagca ttaagtttta
     4801 gttttaaaaa ggtcaatttt ccaaacattc agcaatcata tttaaattta cagctaggaa
     4861 caagagcctt gggtcatgtc ctaccaaaga acataactca atattctaca catgacaatc
     4921 tgaataacct taaagcctct aatcccataa caggccacaa attttggaca gagaactaat
     4981 gatcctcctg agaaaactgg aagaaatcca gggaaaagaa attcctgtgt cctccaaact
     5041 cagaaatctc taattatgtc agtattctct gctttagtcc taggtcagat tgcacacatc
     5101 taaaataacc tcttaaagtt tcctcctagc gacctaaacc attattaata tcaaattaac
     5161 catcaaaaca ctttcctctc aatatgctgc acacaaacct cctcctggaa cctcctccat
     5221 ctggatcctc cccaatcaaa agtataggta tttaacatat aagcaaggaa gtaatgtaaa
     5281 catgaccttg gtcacaaata tgtcatctaa aaacaattta gtcaaggtat ggaggaaatt
     5341 cgagaacctg aatcttttta agtattttga gcacaggaac aattggcaaa aggaatccag
     5401 gtatagacaa aacccagagc ccagagctct gggcgaaaaa tgagttgctg gtgaagacgt
     5461 tacacaagta acatgagaaa gcagaaaatg caggtcatcc acgcacccct gacccaggcc
     5521 agcagggcgg gctgcagcat cagtacacag gagaaagatc cttattccta agaatgagaa
     5581 aggcaaaggc gcccgataga ataaattagc atagaagggg ctttcccagg agttaaaact
     5641 ttccttctga gcgattacct actaaaacca gggcttttgc ccactaccat ttacctagga
     5701 tcttggcttg cacggattca taggggcata tccctccccc tcttctttag agtcgttctt
     5761 aaaagatcgc tctccacgcc ctaggcaggg aaaacgacaa aatctggctc aattccaggc
     5821 tagaacccta caaattcaac agggatatcg caaggatact ggggcatacg ccacagggag
     5881 tccaagaatg tgaggtgggg gtggcgaagg taatgtcttt ggtgtgggaa aagcagcagc
     5941 catctgagat aggaactgga aaaccagagg agaggcgttc aggaagatta tggaggggag
     6001 gactgggccc ccacgagcga ccagagttgt cacaaggccg caagaacagg ggaggtgggg
     6061 ggctcaggga cagaaaaaaa agtatgtgta ttttgagagc agggttggga ggcctctcct
     6121 gaaaagggta taaacgtgga gtaggcaata cccaggcaaa aaggggagac cagagtaggg
     6181 ggaggggaag agtcctgacc cagggaagac attaaaaagg tagtggggtc gactagatga
     6241 aggagagcct ttctctctgg gcaagagcgg tgcaatggtg tgtaaaggta gctgagaaga
     6301 cgaaaagggc aagcatcttc ctgctaccag gctggggagg cccaggccca cgaccccgag
     6361 gagagggaac gcagggagac tgaggtgacc cttctttccc ccggggcccg gtcgtgtggt
     6421 tcggtgtctc ttttctgttg gacccttacc ttgacccagg cgctgccggg gcctgggccc
     6481 gggctgcggc gcacggcact cccgggaggc agcgagactc gagttaggcc caacgcggcg
     6541 ccacggcgtt tcctggccgg gaatggcccg tacccgtgag gtgggggtgg ggggcagaaa
     6601 aggcggagcg agcccgaggc ggggaggggg agggccaggg gcggaggggg ccggcactac
     6661 tgtgttggcg gactggcggg actagggctg cgtgagtctc tgagcgcagc gggcggcggc
     6721 cgcccctccc ccggcggcgg cagcggcggc agcggcggca gctcactcag cccgctgccc
     6781 gagcggaaac gccactgacc gcacggggat tcccagtgcc ggcgccaggg gcacgcggga
     6841 cacgccccct cccgccgcgc cattggcctc tccgcccacc gccccacact tattggccgg
     6901 tgcgccgcca atcagcggag gctgccgggg ccgcctaaag aagaggctgt gctttggggc
     6961 tccggctcct cagagagcct cggctaggta ggggatcggg actctggcgg gagggcggct
     7021 tggtgcgttt gcggggacgc gtaagcttgc ttgttctttt tgcagaagct cagaataaac
     7081 gctcaacttt ggcagatcta ccatggtgag caagggcgag gagctgttca ccggggtggt
     7141 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     7201 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     7261 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tggggcgtgc agtgcttcgc
     7321 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     7381 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     7441 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     7501 ggacggcaac atcctggggc acaagctgga gtacaacgcc atcagcgaca acgtctatat
     7561 caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga
     7621 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     7681 cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa
     7741 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     7801 catggacgag ctgtacaagt aaggatccat aatcagccat accacatttg tagaggtttt
     7861 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     7921 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     7981 aaatttcaca aataaagcat ttttttcact gccccgagct tcctcgctcg cggccgcgaa
     8041 ttccgaagtt cctattctct agaaagtata ggaacttcag gtctgaagag gagtttacgt
     8101 ccagccaagc tagcttggct gcaggtcgtc gaaattctac cgggtagggg aggcgctttt
     8161 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     8221 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     8281 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     8341 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     8401 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     8461 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     8521 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     8581 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc
     8641 gacctgcagc ctgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     8701 aaggtgagga actaaaccat gggatcggcc attgaacaag atggattgca cgcaggttct
     8761 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     8821 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     8881 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     8941 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     9001 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     9061 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     9121 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     9181 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     9241 gccaggctca aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc
     9301 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     9361 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     9421 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     9481 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaggggatca attctctaga
     9541 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     9601 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     9661 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     9721 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     9781 atggcttctg aggcggaaag aaccagctgg ggctcgacta gagcttgcgg aacccttcga
     9841 agaagttcct attctctaga aagtatagga acttcatgca tgtttaaacc gccggcggtt
     9901 aacccgcgga agcttacgcg tctcgagata acttcgtata aagtatccta tacgaagtta
     9961 tggtac
//
