LOCUS       sequence-457514-        6943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses wild-type Cas9 and gRNA for ACTB gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6943)
  TITLE     Assay for Site-specific Homologous Recombination Activity in 
            adherent cells, suspension cells, and tumor tissues
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6943)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCG-ACTB-C4
FEATURES             Location/Qualifiers
     source          1..6943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..274
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     26..46
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     197..216
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        296..371
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        433..644
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      663..672
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             669..4772
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             669..4772
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4773..4820
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4821..4844
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4864..4881)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4870..5094
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(5157..5745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5237..5256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5916..6707,6708..6776))
                     /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     6539..6558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6777..6881)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atccagtttg gttaattaag gtaccgaggg cctatttccc atgattcctt catatttgca
       61 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
      121 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
      181 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
      241 ggctttatat atcttgtgga aaggacgaaa caccgagtcc gcctagaagc atttggtttt
      301 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
      361 cgagtcggtg cttttttgaa ttcgctagct aggtcttgaa aggagtggga attggctccg
      421 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
      481 tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
      541 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
      601 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggaccgg ttctagagcg
      661 ctgccaccat ggacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct
      721 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
      781 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa
      841 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
      901 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
      961 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
     1021 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
     1081 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
     1141 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     1201 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     1261 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     1321 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     1381 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     1441 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     1501 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     1561 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     1621 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     1681 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     1741 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     1801 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     1861 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     1921 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     1981 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     2041 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     2101 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact
     2161 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     2221 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     2281 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
     2341 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     2401 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     2461 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     2521 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     2581 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     2641 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     2701 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     2761 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     2821 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     2881 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     2941 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     3001 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     3061 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     3121 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     3181 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
     3241 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     3301 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     3361 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     3421 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     3481 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     3541 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     3601 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     3661 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     3721 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     3781 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     3841 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     3901 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     3961 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     4021 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     4081 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     4141 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     4201 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     4261 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     4321 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     4381 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     4441 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     4501 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     4561 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     4621 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     4681 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     4741 cacggatcga cctgtctcag ctgggaggcg acaagcgacc tgccgccaca aagaaggctg
     4801 gacaggctaa gaagaagaaa gattacaaag acgatgacga taagggatcc tgacaagctt
     4861 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     4921 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     4981 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     5041 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     5101 aggcggaaag aaccagctgg ggctctagac cgtaaaaagg ccgcgttgct ggcgtttttc
     5161 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5221 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5281 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5341 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5401 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5461 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5521 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5581 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5641 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5701 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5761 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5821 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5881 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5941 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6001 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6061 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6121 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6181 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6241 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6301 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6361 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6421 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6481 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6541 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6601 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6661 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6721 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6781 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6841 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6901 ccacctaaat tgtaagcgtt aactcgagac agggacagca gag
//
