LOCUS       sequence-199283-        7407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  eCas9-1.1 mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7407)
  AUTHORS   Kim S, Bae T, Hwang J, Kim JS
  TITLE     Rescue of high-specificity Cas9 variants using sgRNAs with matched 
            5' nucleotides.
  JOURNAL   Genome Biol. 2017 Nov 15;18(1):218. doi: 10.1186/s13059-017-1355-3.
  PUBMED    29141659
REFERENCE   2  (bases 1 to 7407)
  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: "Genome 
            Biol."; date: "2017-11-15"; volume: "18(1):218. doi"; pages: " 
            10.1186/s13059-017-1355-3"
COMMENT     Sequence Label: p3s-eCas9-1.1
FEATURES             Location/Qualifiers
     source          1..7407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        130..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     664..684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     758..777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        758..776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      790..799
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             802..828
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             802..828
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             838..858
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             886..2601
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     promoter        complement(5020..5038)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5021..5038)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5058..5075)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5064..5288
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5376..5393)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5547..6135)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5627..5646)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6306..7097,7098..7166))
                     /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     6929..6948
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7167..7271)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7346..7365)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
       61 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
      121 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
      181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      541 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      601 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      661 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
      721 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
      781 cccaagcttg ccaccatggt gtacccctac gacgtgcccg actacgccga attgcctcca
      841 aaaaagaaga gaaaggtagg gatccgaatt cccggggaaa aaccggacaa gaagtacagc
      901 atcggcctgg acatcggtac caacagcgtg ggctgggccg tgatcaccga cgagtacaag
      961 gtgcccagca agaagttcaa ggtgctgggc aacaccgacc gccacagcat caagaagaac
     1021 ctgatcggcg ccctgctgtt cgacagcggc gagaccgccg aggccacccg cctgaagcgc
     1081 accgcccgcc gccgctacac ccgccgcaag aaccgcatct gctacctgca ggagatcttc
     1141 agcaacgaga tggccaaggt ggacgacagc ttcttccacc gcctggagga gagcttcctg
     1201 gtggaggagg acaagaagca cgagcgccac cccatcttcg gcaacatcgt ggacgaggtg
     1261 gcctaccacg agaagtaccc caccatctac cacctgcgca agaagctggt ggacagcacc
     1321 gacaaggccg acctgcgcct gatctacctg gccctggccc acatgatcaa gttccgcggc
     1381 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     1441 cagctggtgc agacctacaa ccagctgttc gaggagaacc ccatcaacgc cagcggcgtg
     1501 gacgccaagg ccatcctgag cgcccgcctg agcaagagcc gccgcctgga gaacctgatc
     1561 gcccagctgc ccggcgagaa gaagaacggc ctgttcggca acctgatcgc cctgagcctg
     1621 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggacgccaa gctgcagctg
     1681 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     1741 gccgacctgt tcctggccgc caagaacctg agcgacgcca tcctgctgag cgacatcctg
     1801 cgcgtgaaca ccgagatcac caaggccccc ctgagcgcca gcatgatcaa gcgctacgac
     1861 gagcaccacc aggacctgac cctgctgaag gccctggtgc gccagcagct gcccgagaag
     1921 tacaaggaga tcttcttcga ccagagcaag aacggctacg ccggctacat cgacggcggc
     1981 gccagccagg aggagttcta caagttcatc aagcccatcc tggagaagat ggacggcacc
     2041 gaggagctgc tggtgaagct gaaccgcgag gacctgctgc gcaagcagcg caccttcgac
     2101 aacggcagca tcccccacca gatccacctg ggcgagctgc acgccatcct gcgccgccag
     2161 gaggacttct accccttcct gaaggacaac cgcgagaaga tcgagaagat cctgaccttc
     2221 cgcatcccct actacgtggg ccccctggcc cgcggcaaca gccgcttcgc ctggatgacc
     2281 cgcaagagcg aggagaccat caccccctgg aacttcgagg aggtggtgga caagggcgcc
     2341 agcgcccaga gcttcatcga gcgcatgacc aacttcgaca agaacctgcc caacgagaag
     2401 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtacaacga gctgaccaag
     2461 gtgaagtacg tgaccgaggg catgcgcaag cccgccttcc tgagcggcga gcagaagaag
     2521 gccatcgtgg acctgctgtt caagaccaac cgcaaggtga ccgtgaagca gctgaaggag
     2581 gactacttca agaagatcga gtgcttcgac agcgtggaga tcagcggcgt ggaggaccgc
     2641 ttcaacgcca gcctgggcac ctaccacgac ctgctgaaga tcatcaagga caaggacttc
     2701 ctggacaacg aggagaacga ggacatcctg gaggacatcg tgctgaccct gaccctgttc
     2761 gaggaccgcg agatgatcga ggagcgcctg aagacctacg cccacctgtt cgacgacaag
     2821 gtgatgaagc agctgaagcg ccgccgctac accggctggg gccgcctgag ccgcaagctt
     2881 atcaacggca tccgcgacaa gcagagcggc aagaccatcc tggacttcct gaagagcgac
     2941 ggcttcgcca accgcaactt catgcagctg atccacgacg acagcctgac cttcaaggag
     3001 gacatccaga aggcccaggt gagcggccag ggcgacagcc tgcacgagca catcgccaac
     3061 ctggccggca gccccgccat caagaagggc atcctgcaga ccgtgaaggt ggtggacgag
     3121 ctggtgaagg tgatgggccg ccacaagccc gagaacatcg tgatcgagat ggcccgcgag
     3181 aaccagacca cccagaaggg ccagaagaac agccgcgagc gcatgaagcg catcgaggag
     3241 ggcatcaagg agctgggcag ccagatcctg aaggagcacc ccgtggagaa cacccagctg
     3301 cagaacgaga agctgtacct gtactacctg cagaacggcc gcgacatgta cgtggaccag
     3361 gagctggaca tcaaccgcct gagcgactac gacgtggacc acatcgtgcc ccagagcttc
     3421 ctggccgacg acagcatcga caacaaggtg ctgacccgca gcgacaagaa ccgcggcaag
     3481 agcgacaacg tgcccagcga ggaggtggtg aagaagatga agaactactg gcgccagctg
     3541 ctgaacgcca agctgatcac ccagcgcaag ttcgacaacc tgaccaaggc cgagcgcggc
     3601 ggcctgagcg agctggacaa ggccggcttc atcaagcgcc agctggtgga gacccgccag
     3661 atcaccaagc acgtggccca gatcctggac agccgcatga acaccaagta cgacgagaac
     3721 gacaagctga tccgcgaggt gaaggtgatc accctgaaga gcaagctggt gagcgacttc
     3781 cgcaaggact tccagttcta caaggtgcgc gagatcaaca actaccacca cgcccacgac
     3841 gcctacctga acgccgtggt gggcaccgcc ctgatcaaga agtaccccgc cctggagagc
     3901 gagttcgtgt acggcgacta caaggtgtac gacgtgcgca agatgatcgc caagagcgag
     3961 caggagatcg gcaaggccac cgccaagtac ttcttctaca gcaacatcat gaacttcttc
     4021 aagaccgaga tcaccctggc caacggcgag atccgcaagg cccccctgat cgagaccaac
     4081 ggcgagaccg gcgagatcgt gtgggacaag ggccgcgact tcgccaccgt gcgcaaggtg
     4141 ctgagcatgc cccaggtgaa catcgtgaag aagaccgagg tgcagaccgg cggcttcagc
     4201 aaggagagca tcctgcccaa gcgcaacagc gacaagctga tcgcccgcaa gaaggactgg
     4261 gaccccaaga agtacggcgg cttcgacagc cccaccgtgg cctacagcgt gctggtggtg
     4321 gccaaggtgg agaagggcaa gagcaagaag ctgaagagcg tgaaggagct gctgggcatc
     4381 accatcatgg agcgcagcag cttcgagaag aaccccatcg acttcctgga ggccaagggc
     4441 tacaaggagg tgaagaagga cctgatcatc aagctgccca agtacagcct gttcgagctg
     4501 gagaacggcc gcaagcgcat gctggccagc gccggcgagc tgcagaaggg caacgagctg
     4561 gccctgccca gcaagtacgt gaacttcctg tacctggcca gccactacga gaagctgaag
     4621 ggcagccccg aggacaacga gcagaagcag ctgttcgtgg agcagcacaa gcactacctg
     4681 gacgagatca tcgagcagat cagcgagttc agcaagcgcg tgatcctggc cgacgccaac
     4741 ctggacaagg tgctgagcgc ctacaacaag caccgcgaca agcccatccg cgagcaggcc
     4801 gagaacatca tccacctgtt caccctgacc aacctgggcg cccccgccgc cttcaagtac
     4861 ttcgacacca ccatcgaccg caagcgctac accagcacca aggaggtgct ggacgccacc
     4921 ctgatccacc agagcatcac cggtctgtac gagacccgca tcgacctgag ccagctgggc
     4981 ggcgactaat aactcgagca tgcatctaga gggccctatt ctatagtgtc acctaaatgc
     5041 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     5101 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     5161 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     5221 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     5281 ctctatggct tctgaggcgg aaagaaccag ctgcattaat gaatcggcca acgcgcgggg
     5341 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5401 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5461 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5521 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5581 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5641 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5701 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5761 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5821 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5881 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5941 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6001 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6061 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6121 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6181 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6241 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6301 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6361 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6421 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6481 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6541 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6601 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6661 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6721 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6781 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6841 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6901 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6961 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7021 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7081 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7141 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7201 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7261 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     7321 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     7381 atctgctccc tgcttgtgtg ttggagg
//
