LOCUS       sequence-304499-        4831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Replace donor vector for cherry insertion using the mouse Rosa26 
            guide RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4831)
  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 4831)
  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: pMC_cherry donor_Rosa target (pE002)
FEATURES             Location/Qualifiers
     source          1..4831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(693..710)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    731..764
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             838..1548
                     /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(983..1001)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1251..1270
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(1696..2490)
                     /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             complement(1696..2490)
                     /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(1807..1826)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2417..2436
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(2854..2871)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3024..3110
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3202..3229
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      3363..3951
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3852..3871
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4105..4122
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4137..4277
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4203..4222)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4363..4385
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcttctctca tccgccaaaa cagccaagct ggagaccgtt tgacattacc ctgttatccc
       61 tagatacatt accctgttat cccagatgac ataccctgtt atccctagat gacattaccc
      121 tgttatccca gatgacatta ccctgttatc cctagataca ttaccctgtt atcccagatg
      181 acataccctg ttatccctag atgacattac cctgttatcc cagatgacat taccctgtta
      241 tccctagata cattaccctg ttatcccaga tgacataccc tgttatccct agatgacatt
      301 accctgttat cccagatgac attaccctgt tatccctaga tacattaccc tgttatccca
      361 gatgacatac cctgttatcc ctagatgaca ttaccctgtt atcccagatg acattaccct
      421 gttatcccta gatacattac cctgttatcc cagatgacat accctgttat ccctagatga
      481 cattaccctg ttatcccaga tgacattacc ctgttatccc tagatacatt accctgttat
      541 cccagatgac ataccctgtt atccctagat gacattaccc tgttatccca gatgacatta
      601 ccctgttatc cctagataca ttaccctgtt atcccagatg acataccctg ttatccctag
      661 atgacattac cctgttatcc cagataaact caatgatgat gatgatgatg gtcgagactc
      721 agcggccgcg gtgccagggc gtgcccttgg gctccccggg cgcgactagt gaattcagat
      781 ctgatatctc tagagtcgag ctagcactcc agtctttcta gaagaagggc cacaaccatg
      841 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
      901 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
      961 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1021 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     1081 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1141 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     1201 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctcagacgg ccccgtaatg
     1261 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     1321 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     1381 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     1441 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     1501 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaatg agtcgaccca
     1561 tgggggcccg ccccaactgg ggtaaccttt gagttctctc agttgggggt aatcagcatc
     1621 atgatgtggt accacatcat gatgctgatt ataagaatgc ggccgccaca ctctagtgga
     1681 tctcgagtta ataattcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
     1741 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
     1801 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg
     1861 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
     1921 atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg ctcgccttga gcctggcgaa
     1981 cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc
     2041 ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca
     2101 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc
     2161 ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca
     2221 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
     2281 cagccacgat agccgcgctg cctcgtcttg cagttcattc agggcaccgg acaggtcggt
     2341 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
     2401 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
     2461 acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc
     2521 agagcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt
     2581 gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt
     2641 ccataaaacc gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct
     2701 ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca
     2761 gcaccgtttc tgcggactgg ctttctacgc gctcgagggg gggccaaacg gtctccagct
     2821 tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag
     2881 aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc
     2941 catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc
     3001 gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     3061 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag
     3121 cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa
     3181 ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac
     3241 aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaccaaaatc
     3301 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3361 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3421 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3481 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3541 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3601 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3661 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3721 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3781 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3841 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3901 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3961 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4021 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4081 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4141 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     4201 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     4261 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4321 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4381 agaggttttc accgtcatca ccgaaacgcg cgaggcagca gatcaattcg cgcgcgaagg
     4441 cgaagcggca tgcataatgt gcctgtcaaa tggacgaagc agggattctg caaaccctat
     4501 gctactccgt caagccgtca attgtctgat tcgttaccaa ttatgacaac ttgacggcta
     4561 catcattcac tttttcttca caaccggcac ggaactcgct cgggctggcc ccggtgcatt
     4621 ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga ccgacggtgg
     4681 cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt tggtcctcgc
     4741 gccagcttaa gacgctaatc cctaactgct ggcggaaaag atgtgacaga cgcgacggcg
     4801 acaagcaaac atgctgtgcg acgctggcga t
//
