LOCUS       pLCA.66/2272                6678 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pLCA.66/2272
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6678)
  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. 2011 Feb 14. ():.
  PUBMED    21324933
REFERENCE   2  (bases 1 to 6678)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            //www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6678
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        11..510
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(46..69)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     443..462
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      524..533
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             530..1129
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1029..1049
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1136..2131
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=-Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     polyA_signal    2159..2383
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2429..2476
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2495..3298
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAACVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2558..2577)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3168..3187
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3336..3560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(3626..3659)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
     primer_bind     complement(3735..3754)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3736..3754)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3761..3778)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3761..3777)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3770..3792)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3919..4374
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4006..4025)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4216..4237
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4400..4504
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4505..5365
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4723..4742)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5536..6124
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6025..6044
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6278..6295
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6412..6433
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6448..6478
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6486..6502
                     /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)."
     primer_bind     6491..6513
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6510..6526
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6510..6526
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        6547..6565
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(6623..6656)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="loxP mutant. Cre-mediated recombination occurs in 
                     the 8-bp core sequence (GCATACAT)."
ORIGIN
        1 taattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
       61 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
      121 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
      181 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
      241 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
      301 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
      361 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
      421 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
      481 ttctcctctt cctcatctcc gggcctttcg acctgcagcc aacgccacca tggggaccga
      541 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct
      601 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat
      661 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
      721 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
      781 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
      841 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
      901 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
      961 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     1021 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     1081 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatccatgcc
     1141 cacgctactg cgggtttata tagacggtcc tcacgggatg gggaaaacca ccaccacgca
     1201 actgctggtg gccctgggtt cgcgcgacga tatcgtctac gtacccgagc cgatgactta
     1261 ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc tacaccacac aacaccgcct
     1321 cgaccagggt gagatatcgg ccggggacgc ggcggtggta atgacaagcg cccagataac
     1381 aatgggcatg ccttatgccg tgaccgacgc cgttctggct cctcatatcg ggggggaggc
     1441 tgggagctca catgccccgc ccccggccct caccctcatc ttcgaccgcc atcccatcgc
     1501 cgccctcctg tgctacccgg ccgcgcgata ccttatgggc agcatgaccc cccaggccgt
     1561 gctggcgttc gtggccctca tcccgccgac cttgcccggc acaaacatcg tgttgggggc
     1621 ccttccggag gacagacaca tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct
     1681 tgacctggct atgctggccg cgattcgccg cgtttacggg ctgcttgcca atacggtgcg
     1741 gtatctgcag ggcggcgggt cgtggcggga ggattgggga cagctttcgg ggacggccgt
     1801 gccgccccag ggtgccgagc cccagagcaa cgcgggccca cgaccccata tcggggacac
     1861 gttatttacc ctgtttcggg cccccgagtt gctggccccc aacggcgacc tgtataacgt
     1921 gtttgcctgg gccttggacg tcttggccaa acgcctccgt cccatgcacg tctttatcct
     1981 ggattacgac caatcgcccg ccggctgccg ggacgccctg ctgcaactta cctccgggat
     2041 ggtccagacc cacgtcacca cccccggctc cataccgacg atctgcgacc tggcgcgcac
     2101 gtttgcccgg gagatggggg aggctaactg agctctagag ctcgctgatc agcctcgact
     2161 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2221 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2281 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2341 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2401 accatctggg gctgaattcc tgcagcctgt tgacaattaa tcatcggcat agtatatcgg
     2461 catagtataa tacgacaagg tgaggaacta aaccatggga tcggccattg aacaagatgg
     2521 attgcacgca ggttctccgg ccgcttgcgt ggagaggcta ttcggctatg actgggcaca
     2581 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     2641 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     2701 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     2761 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     2821 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     2881 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     2941 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3001 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgatg atctcgtcgt
     3061 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3121 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3181 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3241 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg
     3301 ggatcaattc tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     3361 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3421 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3481 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3541 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct cgactagagc
     3601 ttgcggaacc cttgaattcc tgcagataac ttcgtataaa gtatcctata ggaagttatg
     3661 gatccaggta ccttcgaaac gcgtcctagg catatgatct agagcggccg ccaccgcggt
     3721 ggagctccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac
     3781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3901 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3961 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4021 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4081 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     4141 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4201 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4261 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4321 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     4381 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4441 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4501 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4561 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4621 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4681 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4741 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4801 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4861 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4921 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4981 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5041 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5101 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5161 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5221 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5281 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5341 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5401 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5461 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5521 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5581 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5641 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     5701 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5761 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5821 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5881 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5941 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6001 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6061 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6121 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6181 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     6241 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     6301 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     6361 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     6421 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     6481 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     6541 gctcgaaatt aaccctcact aaagggaaca aaagctgcgg gccccccctc gaagatctct
     6601 cgaggctagc atgcatatcg attaccgttc gtataatgta tgctatacga agttatacta
     6661 gtaagcttga tatcgaat
//