LOCUS       sequence-326384-        5891 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used as a donor vector for the targeted knock-in of a CD55 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 5891)
  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 5891)
  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-CD55-NeoR
FEATURES             Location/Qualifiers
     source          1..5891
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(952..985)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     intron          1028..1257
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1240..1259
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1302..1355
                     /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             1359..2153
                     /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(1413..1432)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2023..2042
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2175..2309
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2199..2218)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2253..2272
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2406..2439)
                     /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(3130..3146)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3131..3147)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     repeat_region   3154..3294
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3154..3283
                     /label=AAV2 ITR
     rep_origin      3369..3824
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3456..3475)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3666..3687
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3841..3860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3960..3982
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4020..4038)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4106..4210
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4211..4279,4280..5071)
                     /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(4429..4448)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5242..5830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5731..5750
                     /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 tccaccgcgg tggcggccgc ttcaaccatc tggtgtttgg
      181 gggaaatagt atcccttcct tcattcatgc tagaactcta tgtgtatata ttattatata
      241 ggatgtttct tgataggacc atgagtgtca atttattttg aattagacta aatatttatg
      301 tggtaggaat acttattcat tcaaaaattt cacatttaat tagtttgagg ttacctaaaa
      361 taaaacaagt atatattcta aattcgttgc tttttcaaaa cttattataa agagaaatta
      421 gaattaaaga agtctaaggg tcaggcatgg tggctcatac ctgtaatccc agcactttgg
      481 gagacagagg caggtggacc acttgaggtc aggagattga gagtagcctg gccaacatga
      541 tgaaaccctg actctactaa aaatacaaaa actagctggg tgtggtggcg catgcctgta
      601 atcccagctt cttggaaggc tgaggcagga gaatcccttg aacctgggag gcagaggttg
      661 cagtgagctg agattatgtc accgcgctcc agcctgggca acagagcaaa actccatctc
      721 aaaaaaaaaa aaaaaaagtc taaggataag aaatatcaat ggttcagatg atgaatttac
      781 atatatgtgc ttgataattt tttcaaaatt tttacctgag ctttattgag gtataatcga
      841 caaatattat cttaggtgta caacatgatg ttttgatata tgtgcacatt gtgaaatgat
      901 taacacagtc aagctaatta acatatctac cacctcacag ctagtggatc cataacttcg
      961 tatagcatac attatacgaa gttatggaat ttacccaatt cgctgtctgc gagggccagc
     1021 tgttggggtg agtactccct ctcaaaagcg ggcatgactt ctgcgctaag attgtcagtt
     1081 tccaaaaacg aggaggattt gatattcacc tggcccgcgg tgatgccttt gagggtggcc
     1141 gcgtccatct ggtcagaaaa gacaatcttt ttgttgtcaa gcttgaggtg tggcaggctt
     1201 gagatctggc catacacttg agtgacaatg acatccactt tgcctttctc tccacaggtg
     1261 tccactccca ggtccaactg cattccggaa cgcgttgtac agagggcaga ggaagtcttc
     1321 taacatgcgg tgacgtggag gagaatcccg gcccaaccat ggctgaacaa gatggattgc
     1381 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     1441 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     1501 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     1561 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     1621 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     1681 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     1741 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     1801 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     1861 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     1921 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     1981 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     2041 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     2101 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     2161 tctggggttc gggggatcca gacatgataa gatacattga tgagtttgga caaaccacaa
     2221 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     2281 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     2341 aggttcaggg ggaggtgtgg gaggtttttt ctagggggcc ccggcgaggc cgggatgagt
     2401 tgggaataac ttcgtatagc atacattata cgaagttatc caaattttaa ggtaccactg
     2461 tgcatatgta tgaattcgat ctagttacct tctttgtgtg tatgcctgat aatttaattt
     2521 taaaaaatca atttgtattc tattctagag aaatcatgac ctaatccggg agaaatacga
     2581 aatggtcaga ttgatgtacc aggtggcata ttatttggtg caaccatctc cttctcatgt
     2641 aacacagggt aagtttgggc atactaaaac cctgtattta ggaaatgaga aaacaaatta
     2701 ggacttaagg tgagattgtt agtttcatga ctggtattca cagctagtag caattaaaac
     2761 aatccctctc ctcaaagacc cttcatcatg agctcatcac agttaaattt agtaaaatgg
     2821 gggcaagaaa ggaagatttc ttaaaccacc agaattctct aaattctgat ttcagtaaat
     2881 atgtgattca atataaaatt ttaaatgtga ttaataaaaa aatttgagat gatgatactt
     2941 gcagaaatct tcaaatgtta aatactaaac caatctttag ttgacctaaa taatgaattt
     3001 gcatgagttt ttctatatgc aaacaagctg ttacaaaaca gcatgagtta aagacattcc
     3061 tagtgtgaaa gtattctaag ttctttgata agttctctgt atagggagaa ggaactagat
     3121 caagcttatc gataccgtcg acctcgaggc cgcaggaacc cctagtgatg gagttggcca
     3181 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     3241 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     3301 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     3361 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3421 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     3481 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3541 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     3601 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3661 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     3721 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     3781 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     3841 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3901 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3961 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     4021 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     4081 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4141 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4201 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4261 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4321 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4381 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4441 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4501 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4561 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4621 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4681 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4741 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4801 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4861 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4921 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4981 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5041 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5101 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5161 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5221 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5281 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5341 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5401 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5461 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5521 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5581 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5641 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5701 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5761 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5821 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5881 tgctcacatg t
//
