LOCUS       sequence-319288-        4895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used as a donor vector for the targeted knock-in of a mutation in 
            PIGA exon 6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4895)
  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 4895)
  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-PIGAmut
FEATURES             Location/Qualifiers
     source          1..4895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2707..2727)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2707..2725)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2746..2762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2746..2762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2759..2781)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2770..2786
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2794..2800,2801..2818,2819..2824))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2839..2860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2977..2994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3148..3736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3228..3247)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3907..4698,4699..4767))
                     /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     4530..4549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4768..4872)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgctcta gaggctgttg acctgtacaa catgcactgc atgttataac
      721 ttccttgagt tcaaaaggag acggtttaaa ggtctaatat atgagactaa gcacaagtaa
      781 tactcaaatt acaaaatatg aatgttggcc catttgatgt gttctgaaca agtatgttta
      841 aagatcttcg ttttttgata ttttagagct tagtacttag cagtgtttta aaaagcaagc
      901 cacctacttg ctcagcaaaa aacgttattt catttgccag catttgacct atactatcta
      961 ctgaaatttc ccccatctct ttatcttctt ttatttgaaa tccatgaaat gcttctgcat
     1021 gtcctcacca tccatgctca gcaacatgta cagcctcatg tggctgttga aagaggaggc
     1081 tctgtgttcc cagaagtatg ctagattgta gctctctgct tgtggttgat gtctgccaga
     1141 tgccggtgag agcatctcag cttagtcaag gaatcttcaa aggcaaatcc tgcttcagta
     1201 ctgctgaagt cactgctgaa ttaaatattg accctacagt ggtgacccca aaagctgttc
     1261 agactttgcg ggactttgga attctagtag ttggctaaaa ttgatggtga ctccctaggg
     1321 gagaggaact gtggcttatt ctttgtatca cacagcctct agcccggtgt cttgcccaga
     1381 ataagtaccc aataagtttt gaatcaaatg gcaataatta tcgtgattaa gttcattaag
     1441 gatatcatta ctatgacaac aacaacaaaa cacaagcaac ttttgtttgt atttgtttat
     1501 aaaatgttcc tgcaggatcg ggtcattgtt tatcatggga caggtgatgg ggtgatgggt
     1561 gaaagtgctc acacttggct tctctccctc caggtatatg actagatatc agtggaagct
     1621 gtgttgccaa tggacaaacg actggacaga cttatttctc actgcggccc agtaacaggc
     1681 tacatctttg ctttgttggc agttttcaac ttcctcttcc tcattttctt gagatggatg
     1741 actccagatt ctatcattga tgttgcaata gatgccactg ggccacgggg tgcctggact
     1801 aataactatt ctcacagtaa aagagggggt gagaataatg agatatctga aaccaggtag
     1861 aaggaagcct agattgtaag attttaaaca tttgtaatag ttctataaag actatggaaa
     1921 ataaccttgc ttttgggggg tttttgtttt tttagagtta atttagtaag ttatgctacc
     1981 tctatatcat tcaatatttt ctgttgagga aagataaaaa tgtatgcaat tcctgagtgt
     2041 agaaacttct tgcacttatt taaaatttag gagagaacat ttaagccact caggtatgca
     2101 atttttcaga ctactgaaat ccctgtagca gagatgtttt aacattatat tttgagagct
     2161 ttgggtgctg aagggccaaa cgttttctgg gcattttttg gccagttttt aatgtaacac
     2221 cattagacac tcaccagatg tttacaagtt ttctttaggg gaactacaac aattatatga
     2281 actgttttat atcatgttca tatacattta ttaggaatct aaatcatgtc tttgaacatt
     2341 tattaggttc actcagtagg tgttacatgt aattaacagg ttccttgagt aagatagtcc
     2401 atcagttacc agcacatttt gaacccctgc tctgtgtaga atgttgaact agatgcttcc
     2461 cgccattaag gaccaggggt gcattcactc tttgtttacc attcaaatgg cttacttcat
     2521 cataattgtg gttgatatga gatcaatatc caacatgcca aaaatgctca tgccagttaa
     2581 tgccaggaaa aaaatcaccg acacactact agtactttgt tcctgttgta tgcattctcc
     2641 taggtagagc ctccatcttc agttgtgttt gtgctcgagg gggggcccgg tacccagctt
     2701 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     2761 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggga gcataaagtg
     2821 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2881 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2941 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3001 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3061 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3121 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3181 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3241 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3301 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3361 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3421 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3481 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3541 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3601 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3661 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3721 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3781 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3841 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3901 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3961 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4021 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4081 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4141 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4201 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4261 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4321 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4381 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4441 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4501 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4561 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4621 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4681 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4741 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4801 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4861 aggggttccg cgcacatttc cccgaaaagt gccac
//
