LOCUS       sequence-242090-        5868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construction of 1st donor for scarless genome editing with unique 
            guide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5868)
  AUTHORS   Ikeda K, Uchida N, Nishimura T, White J, Martin RM, Nakauchi H, 
            Sebastiano V, Weinberg KI, Porteus MH
  TITLE     Efficient scarless genome editing in human pluripotent stem cells.
  JOURNAL   Nat Methods. 2018 Dec;15(12):1045-1047. doi: 
            10.1038/s41592-018-0212-y. Epub 2018 Nov 30.
  PUBMED    30504872
REFERENCE   2  (bases 1 to 5868)
  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"; doi: 
            "10.1038/s41592-018-0212-y"; journalName: "Nat Methods"; date: 
            "2018-12"; volume: "15"; issue: "12"; pages: "1045-1047"
COMMENT     Sequence Label: pSLHDR03_mCherry_tCD19_unique guide
FEATURES             Location/Qualifiers
     source          1..5868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      153..741
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     642..661
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        complement(865..1113)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     polyA_signal    1199..1423
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1412..1429
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(2459..2512)
                     /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             complement(2513..3220)
                     /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(2788..2807)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3057..3075
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3217..3226
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3398..3417)
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     promoter        4542..4790
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        4885..4989
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4990..5058,5059..5850)
                     /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(5208..5227)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
       61 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
      121 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
      181 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
      241 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
      301 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
      361 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
      421 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
      481 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
      541 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
      601 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
      661 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
      721 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
      781 tgctggcctt ttgctcacat gtgcggcaaa aaagcaagct gaccctgaag ttcggacagc
      841 acacgaactt cagggtcagc ttgcggtgtt tcgtcctttc cacaagatat ataaagccaa
      901 gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa aacataattt
      961 taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat
     1021 ctttgtgttt acagtcaaat taattccaat tatctctcta acagccttgt atcgtatatg
     1081 caaatatgaa ggaatcatgg gaaataggcc ctcattcttg gggttgagct aaggcaggct
     1141 tgggcagagg tggaggtgta gaatcgcgac cgagacatcg atcggagacc gtcgtacgcc
     1201 atagagccca ccgcatccac agcatgcctg ctattgtctt cccaatcctc ccccttgctg
     1261 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
     1321 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
     1381 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagaac
     1441 ttcagggtca gcttgctcag aacctcctgg tggggtcggt catcctcttc ctcttcctcc
     1501 tcagcaccag ggccctctgc aggtgcagga tgcccaccag gctgcacagg cagaagatca
     1561 ggtaggccag ggtcacggcg ctcaccttcc agccgccggt cctcagcagc cagtgccaca
     1621 gcacgggcct ggcggtgatc tccaggtgga agctcatggt caggttgccc ctgtggcagt
     1681 agtacttgcc ggcgtcctgg gcggtggccc tgggcagcag caggccggtc tccatcaccc
     1741 acatgtccct ggcgggcctg tcgtccttca gctccaggct cagcaggctc ttggggccct
     1801 tggggtgcac gtgggtccag ctcagggggc ccctgctcac gctgtcgggg ggcacgccgc
     1861 agctcagcca cagggtgctg ccgggggcca tggtcaggtc ctggctcagg ctctggttca
     1921 ggctgtccct ggggggcagg caggggggct cgccctccca gatctcgggc ctgtccttgg
     1981 cccacacgta cagcttgggg ctcatcagct tgccgctggg gctgctgggg ccctcgctgc
     2041 tcctgttctt caggccgcag cccaggccgc ccaggtcgct cacgttccac ctgaacagct
     2101 cgccgctgcc ctccacgttc acggtccagc cgggctgcca ggccttctcg ctgggggggc
     2161 cgggctggca caggtagaag ccgcccatct gctggctcac gttgaagatg aacagccaga
     2221 tggccagggg cctcatgtgg atgcccaggc cgggcaggcc caggctcagc ttcaggaagg
     2281 gcttcagggg gctctccctg ctccaggtca gctgctgggt ggggccgtcg ctggtgccct
     2341 tcaggcactg cagcacggcg ttgtcgccct cctccacctt caccaccagg ggctcctcgg
     2401 gcctcacctc catgggggtc aggaacagca ggaagaacag cagcctgggg gggggcatgg
     2461 ggccggggtt ctcctccacg tcgccgcagg tcagcaggct gcccctgccc tccttgtaca
     2521 gctcgtccat gccgccggtg gagtggcggc cctcggcgcg ttcgtactgt tccacgatgg
     2581 tgtagtcctc gttgtgggag gtgatgtcca acttgatgtt gacgttgtag gcgccgggca
     2641 gctgcacggg cttcttggcc ttgtaggtgg tcttgacctc agcgtcgtag tggccgccgt
     2701 ccttcagctt cagcctctgc ttgatctcgc ccttcagggc gccgtcctcg gggtacatcc
     2761 gctcggagga ggcctcccag cccatggtct tcttctgcat tacggggccg tcggagggga
     2821 agttggtgcc gcgcagcttc accttgtaga tgaactcgcc gtcctgcagg gaggagtcct
     2881 gggtcacggt caccacgccg ccgtcctcga agttcatcac gcgctcccac ttgaagccct
     2941 cggggaagga cagcttcaag tagtcgggga tgtcggcggg gtgcttcacg taggccttgg
     3001 agccgtacat gaactgaggg gacaggatgt cccaggcgaa gggcaggggg ccacccttgg
     3061 tcaccttcag cttggcggtc tgggtgccct cgtaggggcg gccctcgccc tcgccctcga
     3121 tctcgaactc gtggccgttc acggagccct ccatgtgcac cttgaagcgc atgaactcct
     3181 tgatgatggc catgttatcc tcctcgccct tgctcaccat ggtggcgatg gatccttcga
     3241 agagctcggt accgtctaac aaaaaagcca aaaacggcca gaatttagcg gacaatttac
     3301 tagtctaaca ctgaaaatta catattgacc caaatgatta catttcaaaa ggtgcctaaa
     3361 aaacttcaca aaacacactc gccaaccccg agcgcatagt tcaaaaccgg agcttcagct
     3421 acttaagaag ataggtacat aaaaccgacc aaagaaactg acgcctcact tatccctccc
     3481 ctcaccagag gtccggcgcc tgtcgattca ggagagccta ccctaggccc gaattctgcg
     3541 tcctgcgacg gagaaaagcc taccgcacac ctaccggcag gtggccccac cctgcattat
     3601 aagccaacag aacgggtgac gtcacgacac gacgagggcg cgcgctccca aaggtacggg
     3661 tgcactgccc aacggcaccg ccataactgc cgcccccgca acagacgaca aaccgagttc
     3721 tccagtcagt gacaaacttc acgtcagggt ccccagatgg tgccccagcc catctcaccc
     3781 gaataagagc tttcccgcat tagcgaaggc ctcaagacct tgggttcttg ccgcccacca
     3841 tgccccccac cttgtttcaa cgacctcaca gcccgcctca caagcgtctt ccattcaaga
     3901 ctcgggaaca gccgccattt tgctgcgctc cccccaaccc ccagttcagg gcaaccttgc
     3961 tcgcggaccc agactacagc ccttggcggt ctctccacac gcttccgtcc caccgagcgg
     4021 cccggcggcc acgaaagccc cggccagccc agcagcccgc tactcaccaa gtgacgatca
     4081 cagcgatcca caaacaagaa ccgcgaccca aatcccggct gcgacggaac tagctgtgcc
     4141 acacccggcg cgtccttata taatcatcgg cgttcaccgc cccacggaga tccctccgca
     4201 gaatcgccga gaagggacta cttttcctcg cctgttccgc tctctggaaa gaaaaccagt
     4261 gccctagagt cacccaagtc ccgtcctaaa atgtccttct gctgatactg gggttctaag
     4321 gccgagtctt atgagcagcg ggccgctgtc ctgagcgtcc gggcggaagg atcaggacgc
     4381 tcgctgcgcc cttcgtctga cgtggcagcg ctcgccgtga ggaggggggc gcccgcggga
     4441 ggcgccaaaa cccggcgcgg aaagtgtctc cgatcaacgc ggggtcgcga gtggaagcag
     4501 gatggttaag ttcagaggtg cagtagacag ctgcaggaca tgagggccta tttcccatga
     4561 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     4621 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     4681 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     4741 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gcaagctgac
     4801 cctgaagttc gtgtgctgtc cgaacttcag ggtcagcttg cttttttcga gttaacgtca
     4861 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4921 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4981 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5041 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5101 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5161 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5221 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5281 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5341 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5401 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5461 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5521 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5581 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5641 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5701 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5761 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5821 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttac
//
