LOCUS       sequence-46757-e        5918 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 5918)
  AUTHORS   Perez-Pinera P, Ousterout DG, Brown MT, Gersbach CA
  TITLE     Gene targeting to the ROSA26 locus directed by engineered zinc 
            finger nucleases.
  JOURNAL   Nucleic Acids Res. 2012 Apr 1;40(8):3741-52. Epub 2011 Dec 14.
  PUBMED    22169954
REFERENCE   2  (bases 1 to 5918)
  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"; journalName: "Nucleic 
            Acids Res."; date: "2012-04-1"; volume: "40(8)"; pages: "3741-52. 
            Epub 2011 Dec 14"
COMMENT     Sequence Label: pCMV-RosaR4 KKR mutations
FEATURES             Location/Qualifiers
     source          1..5918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        87..390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        391..594
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     545..565
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     591..615
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        825..843
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     825..842
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     856..875
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             922..948
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             976..996
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1989..1998
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2002..2136
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2175..2309
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        2382..2739
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2382..2573
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2405..2425)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2590..2725
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2759..2776)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2759..2775)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2768..2790)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2988..3443
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3075..3094)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3285..3306
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3460..3479)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3614..3636
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3689..3707)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3775..3879
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4065..4443
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene; 
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     primer_bind     complement(4099..4118)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4912..5500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5401..5420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5654..5671
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5824..5829,5830..5847,5848..5854)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5862..5878
                     /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     5867..5889
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5886..5902
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5886..5902
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgaattga ttattgacag ttattaatag taatcattac ggggtcatta
       61 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      121 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      181 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      241 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      301 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      361 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      421 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      481 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      541 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
      601 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
      661 cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc
      721 aagagtgacg taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa
      781 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
      841 agaataacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc
      901 tcggttctat cgattgacat gtacccatac gatgtcccag actacgcgaa ttcccctggg
      961 atcccaggca tggggcccaa aaagaaacgc aaagttgggc gcctcgagcc cggggagaaa
     1021 ccatacaaat gccccgagtg tggaaagtca tttagtgatc caggcgcatt agtaagacat
     1081 cagcggacac ataccgggga gaaaccttat aaatgcccag aatgcgggaa atcgttcagt
     1141 caaagagcac atttagaaag acatcaacgg acccacaccg gggagaagcc atacaagtgt
     1201 cccgaatgcg ggaagtcatt ctccagaagt gacgatttag taagacatca gcgcacgcac
     1261 accggggaaa agccatataa atgccccgag tgcggcaaat cattcagcag aagcgaccac
     1321 ctgaccaccc accagcgcac ccataccggt gcggccgccc gcgccctggt gaagagcgag
     1381 ctggaggaga agaagtccga gctgcggcac aagctgaagt acgtgcccca cgagtacatc
     1441 gagctgatcg agatcgccag gaaccccacc caggaccgca tcctggagat gaaggtgatg
     1501 gagttcttca tgaaggtgta cggctacagg ggagagcacc tgggcggaag cagaaagcct
     1561 gacggcgcca tctatacagt gggcagcccc atcgattacg gcgtgatcgt ggacacaaag
     1621 gcctacagcg gcggctacaa tctgcctatc ggccaggccg acgagatgca gagatacgtg
     1681 aaggagaacc agacccggaa taagcacatc aaccccaacg agtggtggaa ggtgtaccct
     1741 agcagcgtga ccgagttcaa gttcctgttc gtgagcggcc acttcaaggg caactacaag
     1801 gcccagctga ccaggctgaa ccgcaaaacc aactgcaatg gcgccgtgct gagcgtggag
     1861 gagctgctga tcggcggcga gatgatcaaa gccggcaccc tgacactgga ggaggtgcgg
     1921 cgcaagttca acaacggcga gatcaacttc tgattataac taatctagag tcgacctgca
     1981 gaagcttggc cgccatggcc caacttgttt attgcagctt ataatggtta caaataaagc
     2041 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2101 tccaaactca tcaatgtatc ttatcatgtc tggatcggga attaattcgg cgcagcacca
     2161 tggcctcatg gcccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     2221 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     2281 tcatcaatgt atcttatcat gtctggatcg atcgggaatt aattcggcgc agcaccatgg
     2341 cctgaaataa cctctgaaag aggaacttgg ttaggtacct tctgaggcgg aaagaaccag
     2401 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2461 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2521 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2581 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2641 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     2701 tagtgaggag gcttttttgg aggcctaggc atttgcaaaa agctgttaac agcttggcac
     2761 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     2821 ttgcagcaca tccccccttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     2881 cttcccaaca gttgcgtagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
     2941 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     3001 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3061 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3121 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3181 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     3241 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3301 aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc
     3361 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3421 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     3481 atagttaagc caactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3541 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3601 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3661 gcgaggcagt attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3721 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3781 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3841 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3901 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3961 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4021 gatctcaaca gcggtaagat cctttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4081 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgatgacg ccgggcaaga
     4141 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4201 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4261 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4321 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4381 gaatgaagcc ataccaaacg acgagcgtga caccacgatg ccagcagcaa tggcaacaac
     4441 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4501 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4561 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4621 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4681 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4741 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4801 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4861 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttcagatcc
     4921 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4981 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5041 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5101 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5161 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5221 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5281 actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc
     5341 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5401 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5461 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5521 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5581 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5641 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     5701 gcctctcccc gcgcgttggc cgattcatta atccagctgg cacgacaggt ttcccgactg
     5761 gaaagcgggc agtgagcgca acgcaattaa tgtgagttac ctcactcatt aggcacccca
     5821 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     5881 tcacacagga aacagctatg accatgatta cgaattaa
//
