LOCUS       sequence-495617-        5822 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Expresses nucleus-localized mCherry using a CMV promoter. Includes a
            GFP cassette flanked by BsmbI cut sites to knock in and coexpress 
            user-defined gRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5822)
  AUTHORS   Hurtado JE, Schieferecke AJ, Halperin SO, Guan J, Aidlen D, Schaffer
            DV, Dueber JE
  TITLE     Nickase fidelity drives EvolvR-mediated diversification in mammalian
            cells.
  JOURNAL   Nat Commun. 2025 Apr 19;16(1):3723. doi: 10.1038/s41467-025-58414-0.
  PUBMED    40253348
REFERENCE   2  (bases 1 to 5822)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-04-19"; volume: "16(1):3723. doi"; pages: " 
            10.1038/s41467-025-58414-0"
COMMENT     Sequence Label: p-mCherry
FEATURES             Location/Qualifiers
     source          1..5822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1153..1401
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1153..1173
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1324..1343
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     RBS             1557..1568
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1575..2288
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2300..2371
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2387..2414
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        2428..2503
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2535..2914
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2915..3118
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3069..3089
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3137..3146
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3149..3169
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3194..3898
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3336..3354)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3604..3623
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4022..4041)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4023..4041)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4048..4065)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4048..4064)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4057..4079)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4206..4661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4293..4312)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4503..4524
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4687..4791
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4792..4860,4861..5652)
                     /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(5010..5029)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctcg aaattaaccc
     1021 tcactaaagg gaacaaaagc tggagctcca ccgcggtggc ggccgctctt aaggggtgca
     1081 gcggcctccg cgccgggttt tggcgcctcg gtgagtgagt gtgtgcgtgt tggatccaag
     1141 gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     1201 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     1261 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     1321 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     1381 ttgtggaaag gacgaaacac ctgagacgga aagtgaaacg tgatttcatg cgtcattttg
     1441 aacattttgt aaatcttatt taataatgtg tgcggcaatt cacatttaat ttatgaatgt
     1501 tttcttaaca tcgcggcaac tcaagaaacg gcaggttcgg atcttagcta ctagagaaag
     1561 aggagaaata ctagatgcgt aaaggcgaag agctgttcac tggtgtcgtc cctattctgg
     1621 tggaactgga tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg
     1681 acgcaactaa tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggttc
     1741 cttggccgac tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg
     1801 accatatgaa gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac
     1861 gcacgatttc ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag
     1921 gcgataccct ggtaaaccgc attgagctga aaggcattga ctttaaagag gacggcaata
     1981 tcctgggcca taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata
     2041 aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg
     2101 tgcagctggc tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc
     2161 cagacaatca ctatctgagc acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg
     2221 atcatatggt tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac
     2281 tgtacaaatg accaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     2341 ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg
     2401 tgggcctttc tgcgtttata cgtctcagtt ttagagctag aaatagcaag ttaaaataag
     2461 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc cgcggcctct
     2521 agactcgagg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     2581 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     2641 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     2701 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     2761 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     2821 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     2881 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     2941 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     3001 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     3061 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
     3121 ctggctaact accggtgcca ccatggcccc aaagaagaag cggaaggtcg gtatccacgg
     3181 agtcccagca gccgtgagca agggcgagga ggataacatg gccatcatca aggagttcat
     3241 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     3301 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     3361 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     3421 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     3481 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     3541 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     3601 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     3661 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     3721 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     3781 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     3841 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaaggg
     3901 atccggtatg taatggctga aaactgggac caggctcact aactcagatc ctactaggtt
     3961 taataaacat ctttattttc attacatctg tgtgttggtt ttttgtgtgg tacccaattc
     4021 gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
     4081 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     4141 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     4201 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4261 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4321 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4381 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     4441 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4501 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4561 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4621 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     4681 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4741 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4801 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4861 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4921 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4981 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5041 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5101 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5161 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5221 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5281 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5341 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5401 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5461 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggttc tcgcggtatc
     5521 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5581 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5641 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5701 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5761 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5821 tc
//
