LOCUS       sequence-319273-        6479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used as a donor vector for the targeted knock-in of a PIGA mutation.
            This plasmid carries a neomycin resistance gene cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6479)
  AUTHORS   Hyodo T, Rahman ML, Karnan S, Ito T, Toyoda A, Ota A, Wahiduzzaman 
            M, Tsuzuki S, Okada Y, Hosokawa Y, Konishi H
  TITLE     Tandem Paired Nicking Promotes Precise Genome Editing with Scarce 
            Interference by p53.
  JOURNAL   Cell Rep. 2020 Jan 28;30(4):1195-1207.e7. doi: 
            10.1016/j.celrep.2019.12.064.
  PUBMED    31995758
REFERENCE   2  (bases 1 to 6479)
  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: 
            "10.1016/j.celrep.2019.12"; journalName: "Cell Rep"; date: 
            "2020-01-28- 28"; volume: "30"; issue: "4"; pages: "1195-1207.e7"
COMMENT     Sequence Label: Donor-PIGA-NeoR
FEATURES             Location/Qualifiers
     source          1..6479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     1051..1067
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1051..1067
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(1069..1102)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     intron          1145..1374
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1357..1376
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1414..1467
                     /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             1471..2265
                     /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="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1525..1544)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2135..2154
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2287..2421
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2311..2330)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2365..2384
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2518..2551)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2604..2620)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2605..2621)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     repeat_region   3742..3882
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3742..3871
                     /label=AAV2 ITR
     rep_origin      3957..4412
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4044..4063)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4254..4275
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4429..4448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4548..4570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4608..4626)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4694..4798
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4799..4867,4868..5659)
                     /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(5017..5036)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5830..6418
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6319..6338
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgagc tcgctagcac gcgtgcggcc gctctagagg ctgttgacct
      181 gtacaacatg cactgcatgt tataacttcc ttgagttcaa aaggagacgg tttaaaggtc
      241 taatatatga gactaagcac aagtaatact caaattacaa aatatgaatg ttggcccatt
      301 tgatgtgttc tgaacaagta tgtttaaaga tcttcgtttt ttgatatttt agagcttagt
      361 acttagcagt gttttaaaaa gcaagccacc tacttgctca gcaaaaaacg ttatttcatt
      421 tgccagcatt tgacctatac tatctactga aatttccccc atctctttat cttcttttat
      481 ttgaaatcca tgaaatgctt ctgcatgtcc tcaccatcca tgctcagcaa catgtacagc
      541 ctcatgtggc tgttgaaaga ggaggctctg tgttcccaga agtatgctag attgtagctc
      601 tctgcttgtg gttgatgtct gccagatgcc ggtgagagca tctcagctta gtcaaggaat
      661 cttcaaaggc aaatcctgct tcagtactgc tgaagtcact gctgaattaa atattgaccc
      721 tacagtggtg accccaaaag ctgttcagac tttgcgggac tttggaattc tagtagttgg
      781 ctaaaattga tggtgactcc ctaggggaga ggaactgtgg cttattcttt gtatcacaca
      841 gcctctagcc cggtgtcttg cccagaataa gtacccaata agttttgaat caaatggcaa
      901 taattatcgt gattaagttc attaaggata tcattactat gacaacaaca acaaaacaca
      961 agcaactttt gtttgtattt gtttataaaa tgttcctgca ggatcgggtc attgtttatc
     1021 atgggacagg tgatggggtg atgggtgaaa tctagaacta gtggatccat aacttcgtat
     1081 agcatacatt atacgaagtt atggaattta cccaattcgc tgtctgcgag ggccagctgt
     1141 tggggtgagt actccctctc aaaagcgggc atgacttctg cgctaagatt gtcagtttcc
     1201 aaaaacgagg aggatttgat attcacctgg cccgcggtga tgcctttgag ggtggccgcg
     1261 tccatctggt cagaaaagac aatctttttg ttgtcaagct tgaggtgtgg caggcttgag
     1321 atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc acaggtgtcc
     1381 actcccaggt ccaactgcat tccggactgt acagagggca gaggaagtct tctaacatgc
     1441 ggtgacgtgg aggagaatcc cggcccaacc atggctgaac aagatggatt gcacgcaggt
     1501 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     1561 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     1621 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     1681 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     1741 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     1801 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     1861 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     1921 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     1981 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     2041 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     2101 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     2161 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     2221 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     2281 tcgggggatc cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     2341 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     2401 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     2461 ggggaggtgt gggaggtttt ttctaggggg ccccggcgag gccgggatga gttgggaata
     2521 acttcgtata gcatacatta tacgaagtta tccaaatttt aaggtaccac tgtgcatatg
     2581 tatgaattcg atatcaagct tatcgatacc gtcgacctcg agtgctcaca cttggcttct
     2641 ctccctccag gtatatgact agatatcagt ggaagctgtg ttgccaatgg acaaacgact
     2701 ggacagactt atttctcact gcggcccagt aacaggctac atctttgctt tgttggcagt
     2761 tttcaacttc ctcttcctca ttttcttgag atggatgact ccagattcta tcattgatgt
     2821 tgcaatagat gccactgggc cacggggtgc ctggactaat aactattctc acagtaaaag
     2881 agggggtgag aataatgaga tatctgaaac caggtagaag gaagcctaga ttgtaagatt
     2941 ttaaacattt gtaatagttc tataaagact atggaaaata accttgcttt tggggggttt
     3001 ttgttttttt agagttaatt tagtaagtta tgctacctct atatcattca atattttctg
     3061 ttgaggaaag ataaaaatgt atgcaattcc tgagtgtaga aacttcttgc acttatttaa
     3121 aatttaggag agaacattta agccactcag gtatgcaatt tttcagacta ctgaaatccc
     3181 tgtagcagag atgttttaac attatatttt gagagctttg ggtgctgaag ggccaaacgt
     3241 tttctgggca ttttttggcc agtttttaat gtaacaccat tagacactca ccagatgttt
     3301 acaagttttc tttaggggaa ctacaacaat tatatgaact gttttatatc atgttcatat
     3361 acatttatta ggaatctaaa tcatgtcttt gaacatttat taggttcact cagtaggtgt
     3421 tacatgtaat taacaggttc cttgagtaag atagtccatc agttaccagc acattttgaa
     3481 cccctgctct gtgtagaatg ttgaactaga tgcttcccgc cattaaggac caggggtgca
     3541 ttcactcttt gtttaccatt caaatggctt acttcatcat aattgtggtt gatatgagat
     3601 caatatccaa catgccaaaa atgctcatgc cagttaatgc caggaaaaaa atcaccgaca
     3661 cactactagt actttgttcc tgttgtatgc attctcctag gtagagcctc catcttcagt
     3721 tgtgtttgtg ctcgaggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3781 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     3841 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     3901 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     3961 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4021 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4081 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4141 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4201 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4261 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     4321 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4381 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4441 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4501 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4561 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4621 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4681 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4741 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4801 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4861 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4921 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4981 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5041 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5101 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5161 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5221 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5281 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5341 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5401 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5461 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5521 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5581 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5641 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5701 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5761 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5821 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5881 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5941 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6001 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6061 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6121 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6181 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6241 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6301 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6361 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6421 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
