LOCUS       sequence-304462-        3106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for the replacement of reporter BFP by mcherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3106)
  AUTHORS   Danner E, Lebedin M, de la Rosa K, Kuhn R
  TITLE     A homology independent sequence replacement strategy in human cells 
            using a CRISPR nuclease.
  JOURNAL   Open Biol. 2021 Jan;11(1):200283. doi: 10.1098/rsob.200283. Epub 
            2021 Jan 27.
  PUBMED    33499763
REFERENCE   2  (bases 1 to 3106)
  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: "Open 
            Biol."; date: "2021-01"; volume: "11(1):200283. doi"; pages: " 
            10.1098/rsob.200283. Epub 2021 Jan 27"
COMMENT     Sequence Label: pReplace_donor_mcherry
FEATURES             Location/Qualifiers
     source          1..3106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     240..262
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     254..271
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     255..271
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     282..301
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        282..300
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             305..316
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             412..1122
                     /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(557..575)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     825..844
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1274..1291)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1384..1972)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1464..1483)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2146..2940)
                     /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"
     promoter        complement(2941..3045)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
       61 ataaatcaaa agaatagacc gagatagggt tgagtggccg ctacagggcg ctcccattcg
      121 ccattcaggc tgcgcaactg ttgggaaggg cgtttcggtg cgggcctctt cgctattacg
      181 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
      241 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgac gtaatacgac tcactatagg
      301 gcgaattgaa ggaaggccgt caaggccgca tttaattaaa ctccagtctt tctagaagat
      361 ggggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catggtgagc
      421 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
      481 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
      541 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
      601 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
      661 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
      721 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
      781 atctacaagg tgaagctgcg cggcaccaac ttcccctcag acggccccgt aatgcagaag
      841 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
      901 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
      961 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     1021 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     1081 ggccgccact ccaccggcgg catggacgag ctgtacaagt aatgaactcc agtctttcta
     1141 gaagatggcg atcgatcatc tgggcctcat gggccttcct ttcactgccc gctttccagt
     1201 cgggaaacct gtcgtgccag ctgcattaac atggtcatag ctgtttcctt gcgtattggg
     1261 cgctctccgc ttcctcgctc actgactcgc tgcgctcggt cgttcgggta aagcctgggg
     1321 tgcctaatga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     1381 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     1441 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     1501 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     1561 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     1621 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     1681 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     1741 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     1801 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     1861 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     1921 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     1981 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     2041 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     2101 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttattaga aaaattcatc
     2161 cagcagacga taaaacgcaa tacgctggct atccggtgcc gcaatgccat acagcaccag
     2221 aaaacgatcc gcccattcgc cgcccagttc ttccgcaata tcacgggtgg ccagcgcaat
     2281 atcctgataa cgatccgcca cgcccagacg gccgcaatca ataaagccgc taaaacggcc
     2341 attttccacc ataatgttcg gcaggcacgc atcaccatgg gtcaccacca gatcttcgcc
     2401 atccggcatg ctcgctttca gacgcgcaaa cagctctgcc ggtgccaggc cctgatgttc
     2461 ttcatccaga tcatcctgat ccaccaggcc cgcttccata cgggtacgcg cacgttcaat
     2521 acgatgtttc gcctgatgat caaacggaca ggtcgccggg tccagggtat gcagacgacg
     2581 catggcatcc gccataatgc tcactttttc tgccggcgcc agatggctag acagcagatc
     2641 ctgacccggc acttcgccca gcagcagcca atcacggccc gcttcggtca ccacatccag
     2701 caccgccgca cacggaacac cggtggtggc cagccagctc agacgcgccg cttcatcctg
     2761 cagctcgttc agcgcaccgc tcagatcggt tttcacaaac agcaccggac gaccctgcgc
     2821 gctcagacga aacaccgccg catcagagca gccaatggtc tgctgcgccc aatcatagcc
     2881 aaacagacgt tccacccacg ctgccgggct acccgcatgc aggccatcct gttcaatcat
     2941 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3001 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3061 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgt
//
