LOCUS       sequence-487070-        6708 bp    DNA     circular SYN 24-SEP-2025
DEFINITION  This plasmid facilitates the assessment of MMR proficiency in human 
            living cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6708)
  AUTHORS   Takedachi A, Matsuishi E, Mizusaki S, Nagasawa T, Fujikane R, Hidaka
            M, Iwai S, Kuraoka I
  TITLE     Novel plasmids for the fluorescence-based evaluation of DNA mismatch
            repair in human cells.
  JOURNAL   Mutat Res. 2022 Jan-Jun;824:111779. doi: 
            10.1016/j.mrfmmm.2022.111779. Epub 2022 Apr 9.
  PUBMED    35472567
REFERENCE   2  (bases 1 to 6708)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mutat Res. 
            2022 Jan-Jun;824:111779. doi: 10.1016/j.mrfmmm.2022.111779. Epub 
            2022 Apr 9."
COMMENT     Sequence Label: pBluescript II NLS-MC-EGFP-tdTomato (pBET2)
FEATURES             Location/Qualifiers
     source          1..6708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(53..641)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(133..152)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(812..1603,1604..1672))
                     /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     1435..1454
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1673..1777)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1804..2260
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1891..1910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2101..2122
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2386..2408
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2400..2417
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2401..2417
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2486..2789
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2790..2993
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      3032..3041
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3044..3064
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3092..3094,3095..3805)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3092..3094,3095..3805)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3134..3155)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3395..3414)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3742..3763
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3806..3871
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    3995..4076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4117..4133)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4117..4133)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4130..4152)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4141..4157
                     /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(join(4165..4171,4172..4189,4190..4195))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4210..4231
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4354..4371)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     enhancer        4475..4778
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4779..4982
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      5032..5041
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5038..6468
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYTVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     polyA_signal    6587..6708
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     6678..6697
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 gatatcgtga gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
       61 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
      121 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
      181 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
      241 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
      301 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
      361 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
      421 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
      481 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
      541 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
      601 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
      661 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
      721 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
      781 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
      841 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
      901 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
      961 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     1021 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     1081 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     1141 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     1201 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     1261 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     1321 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     1381 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     1441 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     1501 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     1561 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     1621 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     1681 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     1741 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     1801 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1861 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1921 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1981 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2041 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2101 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2161 tataagggat tttgccgatt tcggcctatt ggttaaaaaa atgagctgat ttaacaaaaa
     2221 tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc
     2281 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     2341 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     2401 gtaaaacgac ggccagtgag cgcgctagtt attaatagta atcaattacg gggtcattag
     2461 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     2521 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     2581 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     2641 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     2701 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     2761 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     2821 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     2881 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     2941 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
     3001 tgaaccgtca gatccgctag cgctaccggt cgccaccatg gggcccaaga agaagcgcaa
     3061 ggtacctcag cggtactagt cacctcagca cgtgagcaag ggcgaggagc tgttcaccgg
     3121 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     3181 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     3241 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     3301 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     3361 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     3421 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     3481 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3541 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3601 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3661 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     3721 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3781 tctcggcatg gacgagctgt acaagtccgg actcagatct cgagctcaag cttcgaattc
     3841 tgcagtcgac ggtaccgcgg gcccgggatc caccggatct agataactga tcataatcag
     3901 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     3961 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     4021 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4081 tagttgtggt ttgtccgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4141 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4201 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4261 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4321 tttgcgtatt gggcggcgcc gaagagccgc ttcctcgctc actgactcgc tgcgctcggt
     4381 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4441 atcaggggat aacgcaggaa agaacatatc gatgcgttac ataacttacg gtaaatggcc
     4501 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     4561 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     4621 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     4681 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     4741 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     4801 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     4861 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     4921 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     4981 ctggtttagt gaaccgtcag atccgctagc gctaccggac tcagatctcg agccaccatg
     5041 gtgagcaagg gcgaggaggt catcaaagag ttcatgcgct tcaaggtgcg catggagggc
     5101 tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     5161 acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc
     5221 ctgtcccccc agttcatgta cggctccaag gcgtacgtga agcaccccgc cgacatcccc
     5281 gattacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     5341 gacggcggtc tggtgaccgt gacccaggac tcctccctgc aggacggcac gctgatctac
     5401 acggtgaaga tgcgcggcac caacttcccc cccgacggcc ccgtaatgca gaagaagacc
     5461 atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag
     5521 atccaccagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagaccatc
     5581 tacatggcca agaagcccgt gcaactgccc ggctactact acgtggacac caagctggac
     5641 atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgctc cgagggccgc
     5701 caccacctgt tcctggggca tggcaccggc agcaccggca gcggcagctc cggcaccgcc
     5761 tcctccgagg acaacaacat ggccgtcatc aaagagttca tgcgcttcaa ggtgcgcatg
     5821 gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     5881 gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg
     5941 gacatcctgt ccccccagtt catgtacggc tccaaggcgt acgtgaagca ccccgccgac
     6001 atccccgatt acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     6061 ttcgaggacg gcggtctggt gaccgtgacc caggactcct ccctgcagga cggcacgctg
     6121 atctacaagg tgaagatgcg cggcaccaac ttcccccccg acggccccgt aatgcagaag
     6181 aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag
     6241 ggcgagatcc accaggccct gaagctgaag gacggcggcc gctacctggt ggagttcaag
     6301 accatctaca tggccaagaa gcccgtgcaa ctgcccggct actactacgt ggacaccaag
     6361 ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga gcgctccgag
     6421 ggccgccacc acctgttcct gtacggcatg gacgagctgt acaagtagga tccgactcta
     6481 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     6541 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     6601 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     6661 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatctta
//
