LOCUS       sequence-274805-        3395 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for PCR-generated donor DNA for rapid tagging of proteins 
            with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3395)
  AUTHORS   Paix A, Rasoloson D, Folkmann A, Seydoux G
  TITLE     Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome
            Engineering using Double-Stranded DNA Donors.
  JOURNAL   Curr Protoc Mol Biol. 2019 Dec;129(1):e102. doi: 10.1002/cpmb.102.
  PUBMED    31710422
REFERENCE   2  (bases 1 to 3395)
  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: "Curr Protoc
            Mol Biol."; date: "2019-12"; volume: "129(1):e102. doi"; pages: " 
            10.1002/cpmb.102"
COMMENT     Sequence Label: pAP1836
FEATURES             Location/Qualifiers
     source          1..3395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..44
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     238..260
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     252..269
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     253..269
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             296..1000
                     /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(438..456)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     706..725
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1048..1064)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1048..1064)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1061..1083)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1072..1088
                     /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(1096..1102,1103..1120,1121..1126))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1141..1162
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1279..1296)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1450..2038)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1530..1549)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2209..3000,3001..3069))
                     /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     2832..2851
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3070..3174)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3242..3260
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3298..3320)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
       61 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc
      121 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
      181 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
      241 agtcacgacg ttgtaaaacg acggccagtg aattcgagct cggtacccgg ggatcgtgag
      301 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
      361 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
      421 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
      481 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
      541 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
      601 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
      661 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
      721 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
      781 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
      841 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
      901 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
      961 gggccgccac tctaccggcg gcatggacga gctgtacaag gatcctctag agtcgacctg
     1021 caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     1081 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     1141 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1201 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1261 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1321 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1381 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1441 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1501 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1561 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1621 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     1681 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1741 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1801 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1861 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     1921 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     1981 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2041 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2101 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     2161 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     2221 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     2281 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     2341 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     2401 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     2461 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     2521 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     2581 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     2641 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     2701 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     2761 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     2821 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     2881 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     2941 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     3001 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     3061 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     3121 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     3181 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     3241 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     3301 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     3361 acaagcccgt cagggcgcgt cagcgggtgt tggcg
//
