LOCUS       sequence-425338-        6987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid to introduce splice donor mutation on the first intron
            of human B2M locus to mediate knock-out.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6987)
  AUTHORS   Zhao Y, Pan Z, Hong Z, Sun M, Hong Y, Peng X, Li X, Wang X, Wang K
  TITLE     Protocol for scarless genome editing of human pluripotent stem cell 
            based on orthogonal selective reporters.
  JOURNAL   STAR Protoc. 2024 May 23;5(2):103084. doi: 
            10.1016/j.xpro.2024.103084.
  PUBMED    38787727
REFERENCE   2  (bases 1 to 6987)
  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: "STAR 
            Protoc."; date: "2024-05-23"; volume: "5(2):103084. doi"; pages: " 
            10.1016/j.xpro.2024.103084"
COMMENT     Sequence Label: B2M-SDMutation-EPG
FEATURES             Location/Qualifiers
     source          1..6987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..31)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    20..244
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   435..497
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     rep_origin      complement(1234..1822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1314..1333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1993..2784,2785..2853))
                     /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     2616..2635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2854..2958)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3026..3044
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     repeat_region   4051..4085
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    4375..4395
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        4396..5574
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4627..5565
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5522..5542
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5584..6180
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5584..5603)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             6187..6243
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(6244..6246,6247..6249,6250..6963)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6244..6246,6247..6249,6250..6960)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6289..6310)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6550..6569)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6897..6918
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
       61 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
      121 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
      181 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
      241 atgggcggcc ctgtccctca taaaagtttt gttactttat agaagaaatt ttgagttttt
      301 gtttttttta ataaataaat aaacataaat aaattgtttg ttgaatttat tattagtatg
      361 taagtgtaaa tataataaaa cttaatatct attcaaatta ataaataaac ctcgatatac
      421 agaccgataa aacacatgcg tcaattttac acatgattat ctttaacgta cgtcacaata
      481 tgattatctt tctagggtta atataagtgg aggcgtcgcg ctggcgggca ttcctgaagc
      541 tgacagcatt cgggccgaga tgtctcgctc cgtggcctta gctgtgctcg cgctactctc
      601 tctttctggc ctggaggcta tccagcgcga gtctctccta ccctcccgct ctggtccttc
      661 ctctcccgct ctgcaccctc tgtggccctc gctgtgctct ctcgctccgt gacttccctt
      721 ctccaagttc tccttggtgg cccgccgtgg ggctagtcca gggctggatc tcggggaagc
      781 ggcggggtgg cctgggagtg gggaaggggg tgcgcacccg ggacgcgcgc tacttgcccc
      841 tttcggcggg gagcagggga gacctttggc ctacggcgac gggagggtcg ggacaaagtt
      901 tagggcgtcg ataagcgtca gagcgccgag gttgggggag ggtttctctt ccgctctttc
      961 gcggggcctc tggctccccc agcgcagctg gagtggggga cgggtaggct cgtcccaaag
     1021 gcgcggcgct gaggtttgtg aacgcgtgga ggggcgcttg gggtctgggg gaggcgtcgc
     1081 ccgggtaagc ctgtctgctg cggctctgct tcccttagac tggagagctg tggacttcgt
     1141 ctaggcgccc gctaagttcg catgtccgaa ttccatgtga gcaaaaggcc agcaaaaggc
     1201 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1261 gcatcacgaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1321 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1381 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1441 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1501 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1561 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1621 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1681 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1741 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1801 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1861 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1921 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     1981 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2041 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2101 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2161 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2221 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2281 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2341 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2401 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2461 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2521 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2581 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2641 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     2701 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     2761 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     2821 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     2881 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2941 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     3001 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcggcgcgg
     3061 gtacccacta aaattgccga gccctttgtc ttccagtgtc taaaatatta atgtcaatgg
     3121 aatcaggcca gagtttgaat tctagtctct tagcctttgt ttcccctgtc cataaaatga
     3181 atgggggtaa ttctttcctc ctacagttta tttatatatt cactaattca ttcattcatc
     3241 catccattcg ttcattcggt ttactgagta cctactatgt gccagcccct gttctagggt
     3301 ggaaactaag agaatgatgt acctagaggg cgctggaagc tctaaagccc tagcagttac
     3361 tgcttttact attagtggtc gtttttttct cccccccgcc ccccgacaaa tcaacagaac
     3421 aaagaaaatt acctaaacag caaggacata gggaggaact tcttggcaca gaactttcca
     3481 aacacttttt cctgaaggga tacaagaagc aagaaaggta ctctttcact aggaccttct
     3541 ctgagctgtc ctcaggatgc ttttgggact atttttctta cccagagaat ggagaaaccc
     3601 tgcagggaat tcccaagctg tagttataaa cagaagttct ccttctgcta ggtagcattc
     3661 aaagatctta atcttctggg tttccgtttt ctcgaatgaa aaatgcaggt ccgagcagtt
     3721 aactggcggg ggcaccatta gcaagtcact tagcatctct ggggccagtc tgcaaagcga
     3781 gggggcagcc ttaatgtgcc tccagcctga agtcctagaa tgagcgcccg gtgtcccaag
     3841 ctggggcgcg caccccagat cggagggcgc cgatgtacag acagcaaact cacccagtct
     3901 agtgcatgcc ttcttaaaca tcacgagact ctaagaaaag gaaactgaaa acgggaaagt
     3961 ccctctctct aacctggcac tgcgtcgctg gcttggagac aggtgacggt ccctgcgggc
     4021 cttgtcctga ttggctgggc acgcgtttaa ccctagaaag atagtctgcg taaaattgac
     4081 gcatgcattc ttgaaatatt gctctctctt tctaaatagc gcgaatccgt cgctgtgcat
     4141 ttaggacatc tcagtcgccg cttggagctc ccgtgaggcg tgcttgtcaa tgcggtaagt
     4201 gtcactgatt ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac
     4261 gtgactttta agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt
     4321 gataacttat tatatatata ttttcttgtt atagatatca gggacagcca tcatcaactt
     4381 tgtatagaaa agttgggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     4441 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     4501 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     4561 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     4621 acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct
     4681 tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg
     4741 gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct
     4801 tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc
     4861 gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg
     4921 acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt
     4981 tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg
     5041 aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg
     5101 cctgctctgg tgcctggtct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg
     5161 gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg
     5221 agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg
     5281 aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg
     5341 tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag
     5401 gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct
     5461 tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt
     5521 ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaaagctt
     5581 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     5641 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     5701 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     5761 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     5821 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     5881 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     5941 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     6001 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     6061 ctggagacct ccgcgcccca caacctcccc ttctacgagc ggctcggctt caccgtcacc
     6121 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     6181 gctagcgcca ctaacttctc cctgttgaaa caagcagggg atgtcgaaga gaatcccggg
     6241 ccaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     6301 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     6361 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     6421 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     6481 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     6541 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     6601 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     6661 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     6721 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     6781 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     6841 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     6901 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     6961 taactcgagt ctagagggcc cgtttaa
//
