LOCUS       Exported                8219 bp ds-DNA     circular SYN 08-JAN-2026
DEFINITION  Mammalian expression of SpCas9-BLM(Nterm).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8219)
  AUTHORS   Wells Burrell
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jan 14, 2026 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8219
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    626..631
                     /label=Kozak
     CDS             632..4735
                     /codon_start=1
                     /label=hSpCas9
                     /note="hSpCas9"
                     /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             4736..4783
                     /codon_start=1
                     /label=NLS
                     /note="NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4784..4894
                     /codon_start=1
                     /label=GS_XTEN_GS_linker
                     /translation="SGGSSGSGSETPGTSESATPESSGGSSSGGGSGGSGS"
     CDS             4895..5476
                     /codon_start=1
                     /label=BLM(Nterm)
                     /translation="AAVPQNNLQEQLERHSARTLNNKLSLSKPKFSGFTFKKKTSSDNN
                     VSVTNVSVAKTPVLRNKDVNVTEDFSFSEPLPNTTNQQRVKDFFKNAPAGQETQRGGSK
                     SLLPDFLQTPKEVVCTTQNTPTVKKSRDTALKKLEFSSSPDSLSTINDWDDMDDFDTSE
                     TSKSFVTPPQSHFVRVSTAQKSKKGKRNFF"
     polyA_signal    5529..5753
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5837..5859)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5848..5864
                     /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        5872..5902
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5917..5938
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(6226..6814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6985..7845)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, 
                     andrelated antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        7846..7950
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8029..8048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgcgccac catggacaag aagtacagca tcggcctgga
      661 catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa
      721 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc
      781 cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag
      841 aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat
      901 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
      961 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga
     1021 gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga
     1081 cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat
     1141 cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca
     1201 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc
     1261 catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc
     1321 cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc
     1381 caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac
     1441 ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt
     1501 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
     1561 cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca
     1621 ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat
     1681 tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga
     1741 agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct
     1801 cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat
     1861 cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta
     1921 cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta
     1981 ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga
     2041 ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag
     2101 cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa
     2161 gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt
     2221 gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga
     2281 cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa
     2341 gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc
     2401 cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga
     2461 ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga
     2521 gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca
     2581 gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat
     2641 ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
     2701 cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa
     2761 agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag
     2821 ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt
     2881 gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
     2941 ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
     3001 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
     3061 gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat
     3121 caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga
     3181 ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt
     3241 gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa
     3301 gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga
     3361 actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca
     3421 cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat
     3481 ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt
     3541 ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa
     3601 cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta
     3661 cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg
     3721 caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat
     3781 taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg
     3841 ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc
     3901 ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat
     3961 cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa
     4021 gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga
     4081 aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga
     4141 aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt
     4201 gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg
     4261 gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc
     4321 caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga
     4381 ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat
     4441 cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt
     4501 gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat
     4561 ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac
     4621 catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca
     4681 gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaagcg
     4741 acctgccgcc acaaagaagg ctggacaggc taagaagaag aaatctggag gatctagcgg
     4801 atctggaagc gagacaccag gaacaagcga gtcagcaaca ccagagagca gtggcggcag
     4861 cagctcggga ggtggttcgg gtggatctgg atcagctgct gttcctcaaa ataatctaca
     4921 ggagcaacta gaacgtcact cagccagaac acttaataat aaattaagtc tttcaaaacc
     4981 aaaattttca ggtttcactt ttaaaaagaa aacatcttca gataacaatg tatctgtaac
     5041 taatgtgtca gtagcaaaaa cacctgtatt aagaaataaa gatgttaatg ttaccgaaga
     5101 cttttccttc agtgaacctc tacccaacac cacaaatcag caaagggtca aggacttctt
     5161 taaaaatgct ccagcaggac aggaaacaca gagaggtgga tcaaaatcat tattgccaga
     5221 tttcttgcag actccgaagg aagttgtatg cactacccaa aacacaccaa ctgtaaagaa
     5281 atcccgggat actgctctca agaaattaga atttagttct tcaccagatt ctttaagtac
     5341 catcaatgat tgggatgata tggatgactt tgatacttct gagacttcaa aatcatttgt
     5401 tacaccaccc caaagtcact ttgtaagagt aagcactgct cagaaatcaa aaaagggtaa
     5461 gagaaacttt ttttaaaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc
     5521 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     5581 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgaga aaattgcatc
     5641 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     5701 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     5761 ggcggaaaga accagctggg gctcgatacc gtcgacctct agctagagct tggcgtaatc
     5821 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5881 agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac tcacattaat
     5941 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6001 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6061 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6121 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6181 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6241 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6301 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6361 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6421 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6481 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6541 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6601 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6661 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6721 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6781 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6841 gtctgacact cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6901 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6961 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7021 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7081 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7141 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7201 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7261 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     7321 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7381 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7441 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7501 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7561 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7621 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7681 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7741 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7801 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7861 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7921 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7981 cgacggatcg ggagatcgat ctcccgatcc cctagggtcg actctcagta caatctgctc
     8041 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     8101 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     8161 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgtt
//
