LOCUS       sequence-429318-        6458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FokI-mCherry-LacI-NLS in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6458)
  AUTHORS   Rona G, Miwatani-Minter B, Zhang Q, Goldberg HV, Kerzhnerman MA, 
            Howard JB, Simoneschi D, Lane E, Hobbs JW, Sassani E, Wang AA, 
            Keegan S, Laverty DJ, Piett CG, Pongor LS, Xu ML, Andrade J, Thomas 
            A, Sicinski P, Askenazi M, Ueberheide B, Fenyo D, Nagel ZD, Pagano M
  TITLE     CDK-independent role of D-type cyclins in regulating DNA mismatch 
            repair.
  JOURNAL   Mol Cell. 2024 Feb 29:S1097-2765(24)00129-1. doi: 
            10.1016/j.molcel.2024.02.010.
  PUBMED    38458201
REFERENCE   2  (bases 1 to 6458)
  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: "Mol Cell. 
            2024 Feb 29:S1097-2765(24)00129-1. doi: 
            10.1016/j.molcel.2024.02.010."
COMMENT     Sequence Label: FokI-mCherry-LacI-NLS
FEATURES             Location/Qualifiers
     source          1..6458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      143..278
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             327..1121
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             327..1121
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(381..400)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     991..1010
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1309..1328)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1353..1400
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1729..2317
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2218..2237
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2492..2795
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2796..2999
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2950..2970
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3028..3037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3040..3627
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     regulatory      3646..3655
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3652..4359
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3797..3815)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4065..4084
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4403..4422)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5464..5484
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5503..5547
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     polyA_signal    5677..5798
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5714..5733)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5768..5787
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5805..6260)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5942..5963)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6154..6173
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6287..6391
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6393..6458,1..292)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        join(6393..6458,1..126)
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6416..6436)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
       61 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
      121 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
      181 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
      241 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
      301 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
      361 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
      421 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
      481 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
      541 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
      601 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
      661 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
      721 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
      781 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
      841 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
      901 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
      961 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     1021 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     1081 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     1141 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     1201 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     1261 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     1321 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     1381 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     1441 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     1501 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     1561 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     1621 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     1681 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     1741 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1801 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1861 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1921 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1981 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2041 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     2101 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     2161 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     2221 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     2281 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2341 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     2401 ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt
     2461 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     2521 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     2581 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     2641 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     2701 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     2761 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     2821 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     2881 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     2941 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
     3001 gtttagtgaa ccgtcagatc cgctagcgcc accatggggc aactagtcaa aagtgaactg
     3061 gaggagaaga aatctgaact tcgtcataaa ttgaaatatg tgcctcatga atatattgaa
     3121 ttaattgaaa ttgccagaaa ttccactcag gatagaattc ttgaaatgaa ggtaatggaa
     3181 ttttttatga aagtttatgg atatagaggt aaacatttgg gtggatcaag gaaaccggac
     3241 ggagcaattt atactgtcgg atctcctatt gattacggtg tgatcgtgga tactaaagct
     3301 tatagcggag gttataatct gccaattggc caagcagatg aaatgcaacg atatgtcgaa
     3361 gaaaatcaaa cacgaaacaa acatatcaac cctaatgaat ggtggaaagt ctatccatct
     3421 tctgtaacgg aatttaagtt tttatttgtg agtggtcact ttaaaggaaa ctacaaagct
     3481 cagcttacac gattaaatca tatcactaat tgtaatggag ctgttcttag tgtagaagag
     3541 cttttaattg gtggagaaat gattaaagcc ggcacattaa ccttagagga agtgagacgg
     3601 aaatttaata acggcgagat aaacttttca ggaggcggac cggtcgccac catggtgagc
     3661 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
     3721 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     3781 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     3841 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
     3901 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     3961 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     4021 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     4081 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     4141 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     4201 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     4261 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     4321 ggccgccact ccaccggcgg catggacgag ctgtacaagt ccggactcag atctcgaggg
     4381 atggtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     4441 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     4501 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     4561 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     4621 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     4681 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     4741 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     4801 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     4861 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     4921 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     4981 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     5041 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     5101 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     5161 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     5221 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     5281 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     5341 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     5401 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgaag cagcctgagg
     5461 cctcctaaga agaagaggaa ggttggtacc gcgggcgctg gtggcctgaa tgacatcttt
     5521 gaggcccaga agatcgagtg gcatgaggga tccaccggat ctagataact gatcataatc
     5581 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     5641 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     5701 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5761 tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa
     5821 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5881 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5941 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     6001 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     6061 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     6121 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     6181 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     6241 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6301 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6361 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     6421 gtgtgtcagt tagggtgtgg aaagtcccca ggctcccc
//
