LOCUS       sequence-410543-        5894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for Frame 2 with mCherry tag and triple Puromycin 
            resistant marker (3xPuroR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5894)
  AUTHORS   Kim J, Kratz AF, Chen S, Sheng J, Kim HK, Zhang L, Singh BK, Chavez 
            A
  TITLE     High-throughput tagging of endogenous loci for rapid 
            characterization of protein function.
  JOURNAL   Sci Adv. 2024 May 3;10(18):eadg8771. doi: 10.1126/sciadv.adg8771. 
            Epub 2024 May 1.
  PUBMED    38691600
REFERENCE   2  (bases 1 to 5894)
  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: "Sci Adv."; 
            date: "2024-05-3"; volume: "10(18):eadg8771. doi"; pages: " 
            10.1126/sciadv.adg8771. Epub 2024 May 1"
COMMENT     Sequence Label: pDNR-mCherry-T2A-3xPuroR-Frame 2
FEATURES             Location/Qualifiers
     source          1..5894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    166..300
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    420..554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    674..808
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             909..1613
                     /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(1051..1069)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1319..1338
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1623..1676
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2274..2327
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2925..2978
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     polyA_signal    3602..3723
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3971..3988)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4142..4730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4222..4241)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4930..5721,5722..5790))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 cgcgatgaat aaatgaaagc ttgcagatct gcgactctag aggatctgcg actctagagg
       61 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
      121 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
      181 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
      241 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
      301 tgcgactcta gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa
      361 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
      421 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
      481 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
      541 atcatgtctg gatctgcgac tctagaggat cataatcagc cataccacat ttgtagaggt
      601 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
      661 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
      721 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
      781 catcaatgta tcttatcatg tctggatccc catcaagctg atccggaact aactaagtaa
      841 gctagcgaat cgactcgaac ttcgtgtgcg agtctggcgg gtctggtggc agtggagggg
      901 gatccctggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
      961 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     1021 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     1081 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     1141 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     1201 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     1261 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     1321 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     1381 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     1441 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     1501 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     1561 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtccggag
     1621 gagagggcag aggaagtctg ctaacatgcg gtgacgtaga agaaaaccca gggcctctga
     1681 ctgaatacaa gcctactgtc aggttggcta caagagacga cgttcctaga gccgtgagaa
     1741 ctctggctgc agccttcgcc gactaccccg ccacgagaca caccgttgac ccagatcggc
     1801 atattgagag agtgactgaa ctgcaggagc tgtttcttac aagagttggc ctcgacatag
     1861 gcaaggtgtg ggtggcggac gacggcgccg ccgtggccgt ctggaccact cccgaatcag
     1921 ttgaggctgg cgccgtattc gctgagatcg gcccgagaat ggctgagctc agcgggagta
     1981 ggctcgcggc acagcagcaa atggagggac tgctggcacc acacaggccc aaagaacccg
     2041 cctggttcct ggcaaccgtc ggtgtatctc ccgatcatca ggggaaaggt ctgggctctg
     2101 ccgtagtgct ccctggcgtg gaggcagctg agagagcagg agtacctgcc ttcttggaga
     2161 cctccgctcc aaggaatctt cccttctatg aacggttggg cttcaccgtg acagccgacg
     2221 tggaagtccc cgaaggcccc cgcacttggt gcatgacgag gaagcctgga gcggaaggta
     2281 gaggttcttt gcttacctgc ggggacgtgg aagagaaccc aggcccactg actgaataca
     2341 agcctactgt caggttggct acaagagacg acgttcctag agccgtgaga actctggctg
     2401 cagccttcgc cgactacccc gccacgagac acaccgttga cccagatcgg catattgaga
     2461 gagtgactga actgcaggag ctgtttctta caagagttgg cctcgacata ggcaaggtgt
     2521 gggtggcgga cgacggcgcc gccgtggccg tctggaccac tcccgaatca gttgaggctg
     2581 gcgccgtatt cgctgagatc ggcccgagaa tggctgagct cagcgggagt aggctcgcgg
     2641 cacagcagca aatggaggga ctgctggcac cacacaggcc caaagaaccc gcctggttcc
     2701 tggcaaccgt cggtgtatct cccgatcatc aggggaaagg tctgggctct gccgtagtgc
     2761 tccctggcgt ggaggcagct gagagagcag gagtacctgc cttcttggag acctccgctc
     2821 caaggaatct tcccttctat gaacggttgg gcttcaccgt gacagccgac gtggaagtcc
     2881 ccgaaggccc ccgcacttgg tgcatgacga ggaagcctgg agcggagggc cgagggtcac
     2941 tccttacttg tggtgatgtc gaggagaatc ctggcccact gactgaatac aagcctactg
     3001 tcaggttggc tacaagagac gacgttccta gagccgtgag aactctggct gcagccttcg
     3061 ccgactaccc cgccacgaga cacaccgttg acccagatcg gcatattgag agagtgactg
     3121 aactgcagga gctgtttctt acaagagttg gcctcgacat aggcaaggtg tgggtggcgg
     3181 acgacggcgc cgccgtggcc gtctggacca ctcccgaatc agttgaggct ggcgccgtat
     3241 tcgctgagat cggcccgaga atggctgagc tcagcgggag taggctcgcg gcacagcagc
     3301 aaatggaggg actgctggca ccacacaggc ccaaagaacc cgcctggttc ctggcaaccg
     3361 tcggtgtatc tcccgatcat caggggaaag gtctgggctc tgccgtagtg ctccctggcg
     3421 tggaggcagc tgagagagca ggagtacctg ccttcttgga gacctccgct ccaaggaatc
     3481 ttcccttcta tgaacggttg ggcttcaccg tgacagccga cgtggaagtc cccgaaggcc
     3541 cccgcacttg gtgcatgacg aggaagcctg gagcgtgagg gcccgcttcg agcagacatg
     3601 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     3661 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     3721 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     3781 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaagatcc gtcgaccgat
     3841 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     3901 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     3961 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4021 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4081 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4141 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4201 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4261 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4321 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4381 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4441 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4501 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4561 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4621 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4681 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4741 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4801 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4861 aatcaatcta aagtatatat gagtaaactt ggtctgacag cggccgcaaa tgctaaacca
     4921 ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct gcctatttcg
     4981 ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg agggcttacc
     5041 atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc ccgatttgtc
     5101 agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta ctttgtccgc
     5161 ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc cagtgagtag
     5221 tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt cgttcggtat
     5281 ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac ccatattatg
     5341 aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt tggccgcggt
     5401 gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc catccgtaag
     5461 atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt gtatacggcg
     5521 accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata gcagtacttt
     5581 gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga tcttgccgct
     5641 attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag catcttttac
     5701 tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa agaagggaat
     5761 gagtgcgaca cgaaaatgtt ggatgctcat actcgtcctt tttcaatatt attgaagcat
     5821 ttatcagggt tactagtacg tctctcaagg ataagtaagt aatattaagg tacgggaggt
     5881 attggacagg ccgc
//
