LOCUS       sequence-57070-e        5407 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses human codon optimized Cas9 D10A mutant which functions as 
            a nickase for genome engineering.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5407)
  AUTHORS   Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville JE,
            Church GM
  TITLE     RNA-Guided Human Genome Engineering via Cas9.
  JOURNAL   Science. 2013 Feb 15;339(6121):823-6. doi: 10.1126/science.1232033
  PUBMED    23287722
REFERENCE   2  (bases 1 to 5407)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2013-02-15- 15"; volume: "339"; 
            issue: "6121"; pages: "823-6"
COMMENT     Sequence Label: hCas9_D10A
FEATURES             Location/Qualifiers
     source          1..5407
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(793..812)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    837..885
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1087..1515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1174..1193)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1384..1405
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1524..1544)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1529..1858
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1709..1844
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1771..1790
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1925..2719
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1979..1998)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2589..2608
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2895..3016
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2932..2951)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2986..3005
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3065..3081)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3065..3081)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3078..3100)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3089..3105
                     /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(3113..3119,3120..3137,3138..3143))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3158..3179
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3296..3313)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3467..4055)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3547..3566)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4226..5017,5018..5086))
                     /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     4849..4868
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5087..5191)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5266..5285)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct taagggttcg atccctaccg gttagtaatg agtttaaacg
      781 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
      841 aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc
      901 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
      961 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     1021 ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg cagctggggc tctagggggt
     1081 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1141 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1201 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc
     1261 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1321 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1381 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1441 atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1501 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     1561 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     1621 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     1681 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     1741 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     1801 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     1861 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     1921 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     1981 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     2041 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     2101 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     2161 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     2221 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     2281 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     2341 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     2401 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     2461 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     2521 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     2581 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     2641 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     2701 tcttgacgag ttcttctgag cgggactctg gggttcgcga aatgaccgac caagcgacgc
     2761 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     2821 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     2881 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     2941 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3001 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     3061 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     3121 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     3181 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3241 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3301 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3361 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3421 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3481 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3541 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3601 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3661 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3721 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3781 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3841 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3901 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3961 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4021 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4081 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4141 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4201 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4261 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4321 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4381 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4441 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4501 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4561 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4621 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4681 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4741 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4801 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4861 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4921 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4981 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5041 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5101 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5161 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     5221 tcgacggatc gggagatctc ccgatcccct atggtcgact ctcagtacaa tctgctctga
     5281 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     5341 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     5401 gcttagg
//
