LOCUS       sequence-318924-        6678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Jul;4(4):537-47. doi: 10.1242/dmm.006569. Epub 
            2011 Feb 14.
  PUBMED    21324933
REFERENCE   2  (bases 1 to 6678)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1242/dmm.006569"; journalName: "Dis Model Mech"; date: 
            "2011-07"; volume: "4"; issue: "4"; pages: "537-47"
COMMENT     Sequence Label: pLCA.66/2272
FEATURES             Location/Qualifiers
     source          1..6678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..500
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(36..59)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     433..452
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      514..523
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             520..1119
                     /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     1019..1039
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1126..2121
                     /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    2149..2373
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2419..2466
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2485..3288
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2548..2567)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3158..3177
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3326..3550
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3616..3649
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /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)."
     primer_bind     complement(3725..3744)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3726..3744)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3751..3768)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3751..3767)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3760..3782)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3909..4364
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3996..4015)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4206..4227
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4390..4494
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4495..4563,4564..5355)
                     /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(4713..4732)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5526..6114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6015..6034
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6268..6285
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6402..6423
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6438..6443,6444..6461,6462..6468)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6476..6492
                     /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     6481..6503
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6500..6516
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6500..6516
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        6537..6555
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    6613..6646
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
       61 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
      121 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
      181 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
      241 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
      301 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
      361 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
      421 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
      481 cctcatctcc gggcctttcg acctgcagcc aacgccacca tggggaccga gtacaagccc
      541 acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg
      601 ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc
      661 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
      721 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
      781 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
      841 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
      901 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
      961 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     1021 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     1081 ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatccatgcc cacgctactg
     1141 cgggtttata tagacggtcc tcacgggatg gggaaaacca ccaccacgca actgctggtg
     1201 gccctgggtt cgcgcgacga tatcgtctac gtacccgagc cgatgactta ctggcaggtg
     1261 ctgggggctt ccgagacaat cgcgaacatc tacaccacac aacaccgcct cgaccagggt
     1321 gagatatcgg ccggggacgc ggcggtggta atgacaagcg cccagataac aatgggcatg
     1381 ccttatgccg tgaccgacgc cgttctggct cctcatatcg ggggggaggc tgggagctca
     1441 catgccccgc ccccggccct caccctcatc ttcgaccgcc atcccatcgc cgccctcctg
     1501 tgctacccgg ccgcgcgata ccttatgggc agcatgaccc cccaggccgt gctggcgttc
     1561 gtggccctca tcccgccgac cttgcccggc acaaacatcg tgttgggggc ccttccggag
     1621 gacagacaca tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct tgacctggct
     1681 atgctggccg cgattcgccg cgtttacggg ctgcttgcca atacggtgcg gtatctgcag
     1741 ggcggcgggt cgtggcggga ggattgggga cagctttcgg ggacggccgt gccgccccag
     1801 ggtgccgagc cccagagcaa cgcgggccca cgaccccata tcggggacac gttatttacc
     1861 ctgtttcggg cccccgagtt gctggccccc aacggcgacc tgtacaacgt gtttgcctgg
     1921 gccttggacg tcttggccaa acgcctccgt cccatgcacg tctttatcct ggattacgac
     1981 caatcgcccg ccggctgccg ggacgccctg ctgcaactta cctccgggat ggtccagacc
     2041 cacgtcacca cccccggctc cataccgacg atctgcgacc tggcgcgcac gtttgcccgg
     2101 gagatggggg aggctaactg agctctagag ctcgctgatc agcctcgact gtgccttcta
     2161 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2221 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2281 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2341 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accatctggg
     2401 gctgaattcc tgcagcctgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     2461 tacgacaagg tgaggaacta aaccatggga tcggccattg aacaagatgg attgcacgca
     2521 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     2581 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     2641 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     2701 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     2761 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     2821 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     2881 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     2941 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3001 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgatg atctcgtcgt gacccatggc
     3061 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3121 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3181 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3241 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg ggatcaattc
     3301 tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3361 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3421 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3481 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3541 ggctctatgg cttctgaggc ggaaagaacc agctggggct cgactagagc ttgcggaacc
     3601 cttgaattcc tgcagataac ttcgtataaa gtatcctata cgaagttatg gatccaggta
     3661 ccttcgaaac gcgtcctagg catatgatct agagcggccg ccaccgcggt ggagctccaa
     3721 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg
     3781 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     3841 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     3901 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3961 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4021 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4081 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4141 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4201 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4261 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4321 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     4381 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4441 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4501 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4561 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4621 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4681 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4741 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4801 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4861 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4921 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4981 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5041 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5101 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5161 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5221 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5281 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5341 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5401 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5461 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5521 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5581 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5641 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5701 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5761 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5821 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5881 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5941 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6001 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6061 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6121 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6181 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     6241 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     6301 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     6361 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     6421 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     6481 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcgaaatt
     6541 aaccctcact aaagggaaca aaagctgcgg gccccccctc gaagatctct cgaggctagc
     6601 atgcatatcg attaccgttc gtataatgta tgctatacga agttatacta gtaagcttga
     6661 tatcgaatta attctacc
//
