LOCUS       sequence-242085-        5503 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construction of 1st donor for scarless genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5503)
  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 5503)
  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: pSLHDR02_EGFP_tCD8
FEATURES             Location/Qualifiers
     source          1..5503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(56..847,848..916))
                     /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     679..698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(917..1021)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(1116..1364)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2489..2508
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      2680..2689
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2686..2688,2689..2691,2692..3402)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2731..2752)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2992..3011)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3339..3360
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3412..3465
                     /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"
     primer_bind     complement(4112..4129)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4118..4342
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4428..4676
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     rep_origin      complement(4800..5388)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4880..4899)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
       61 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
      121 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
      181 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
      241 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
      301 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
      361 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
      421 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
      481 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
      541 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
      601 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
      661 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
      721 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
      781 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
      841 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
      901 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
      961 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     1021 gcacatttcc ccgaaaagtg ccacctgacg ttaactcgaa aaaagcaagc tgaccctgaa
     1081 gttcggacag cacacgaact tcagggtcag cttgcggtgt ttcgtccttt ccacaagata
     1141 tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta
     1201 aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt
     1261 tgtactaata tctttgtgtt tacagtcaaa ttaattccaa ttatctctct aacagccttg
     1321 tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctcatgtcc tgcagctgtc
     1381 tactgcacct ctgaacttaa ccatcctgct tccactcgcg accatcgatg agcggcagtg
     1441 cgcttccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
     1501 ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
     1561 cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
     1621 gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
     1681 aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
     1741 gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt
     1801 cgcggttctt gtttgtggat cgctgtgatc gtcacttggt gagtagcggg ctgctgggct
     1861 ggccggggct ttcgtggccg ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc
     1921 caagggctgt agtctgggtc cgcgagcaag gttgccctga actgggggtt ggggggagcg
     1981 cagcaaaatg gcggctgttc ccgagtcttg aatggaagac gcttgtgagg cgggctgtga
     2041 ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga acccaaggtc ttgaggcctt
     2101 cgctaatgcg ggaaagctct tattcgggtg agatgggctg gggcaccatc tggggaccct
     2161 gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt
     2221 tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc
     2281 gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg
     2341 cggtaggctt ttctccgtcg caggacgcag aattcgggcc tagggtaggc tctcctgaat
     2401 cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg
     2461 gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg
     2521 ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca
     2581 atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta aattctggcc gtttttggct
     2641 tttttgttag acggtaccga gctcttcgaa ggatccatcg ccaccatggt gtctaagggc
     2701 gaagagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     2761 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2821 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     2881 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     2941 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     3001 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     3061 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     3121 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     3181 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     3241 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     3301 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     3361 accgccgccg ggatcactct cggcatggac gagctgtaca agggatccgg agaggggaga
     3421 ggatcactgc tgacttgcgg ggatgtggaa gagaacccag ggcccatggc tctgcctgtg
     3481 acagcactgc tgctgccact ggccctgctg ctgcacgcag ctcgaccatc ccagttcaga
     3541 gtctctcctc tggaccgcac atggaacctg ggcgagactg tggaactgaa gtgccaggtc
     3601 ctgctgtcta atcctacttc tggctgtagt tggctgttcc agccacgagg agcagcagct
     3661 agtcccacct ttctgctgta cctgtcacag aacaagccaa aagcagccga gggactggat
     3721 actcagaggt tttctgggaa gcgcctggga gacaccttcg tgctgacact gagtgatttt
     3781 cggagagaga acgaaggcta ctatttctgc agcgccctga gcaattccat catgtatttc
     3841 tcccattttg tgcccgtctt tctgccagcc aaacccacca caactcctgc tccaagaccc
     3901 cctactcccg cacctaccat tgcctcacag cctctgagcc tgcgaccaga agcatgtagg
     3961 ccagctgcag gaggagcagt gcacacaagg ggcctggact tcgcctgcga tatctacatt
     4021 tgggcacctc tggctggaac ctgtggcgtg ctgctgctga gcctggtcat cacactgtat
     4081 tgcaaccatt gaggcgcgcc ccgctgatca gcctcgactg tgccttctag ttgccagcca
     4141 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4201 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4261 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4321 ggggatgcgg tgggctctat ggcgtacgct gccgctcatc gcagggtatg gtcgcgattc
     4381 tacacctcca cctctgccca agcctgcctt agctcaaccc caagaatgag ggcctatttc
     4441 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     4501 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     4561 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     4621 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcaa
     4681 gctgaccctg aagttcgtgt gctgtccgaa cttcagggtc agcttgcttt tttgccgcac
     4741 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4801 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4861 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4921 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4981 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5041 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5101 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5161 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5221 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5281 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5341 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5401 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5461 atgagattat caaaaaggat cttcacctag atccttttaa att
//
