LOCUS       Exported                6924 bp ds-DNA     circular SYN 03-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pJIT163-Ubi-2NLS-rCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6924)
  AUTHORS   Rising star
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Apr 3, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6924
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        13..32
                     /label=T7
     misc_feature    33..128
                     /label=DEN2
     CDS             129..4331
                     /codon_start=1
                     /label=zCas9
                     /translation="MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPS
                     KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSN
                     EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD
                     KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV
                     DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ
                     LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKR
                     YDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKM
                     DGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
                     ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN
                     LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT
                     VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI
                     VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKT
                     ILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI
                     LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL
                     KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK
                     VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA
                     GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY
                     KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA
                     TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQ
                     VNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE
                     KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG
                     RKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE
                     IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF
                     DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK"
     terminator      4332
                     /label=E9ter
     misc_feature    4338..4583
                     /label=M-ZmUBI-3UTR
     misc_feature    4704..4709
                     /label=BsaI
     primer_bind     complement(4772..4788)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4796..4812
                     /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(4820..4850)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4865..4886
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5174..5762)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5933..6793)
                     /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     6757..6776
                     /label=CX-Amp-F
     promoter        complement(6794..6898)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagctcggta cctaatacga ctcactatag ggagttgtta gtctacgtgg accgacaaag
       61 acagattctt tgagggagct aagctcaacg tagttctaac agttttttaa ttagagagca
      121 gatctctgat ggctccgaag aagaagagga aggttggcat ccacggggtg ccagctgctg
      181 acaagaagta ctcgatcggc ctcgatattg ggactaactc tgttggctgg gccgtgatca
      241 ccgacgagta caaggtgccc tcaaagaagt tcaaggtcct gggcaacacc gatcggcatt
      301 ccatcaagaa gaatctcatt ggcgctctcc tgttcgacag cggcgagacg gctgaggcta
      361 cgcggctcaa gcgcaccgcc cgcaggcggt acacgcgcag gaagaatcgc atctgctacc
      421 tgcaggagat tttctccaac gagatggcga aggttgacga ttctttcttc cacaggctgg
      481 aggagtcatt cctcgtggag gaggataaga agcacgagcg gcatccaatc ttcggcaaca
      541 ttgtcgacga ggttgcctac cacgagaagt accctacgat ctaccatctg cggaagaagc
      601 tcgtggactc cacagataag gcggacctcc gcctgatcta cctcgctctg gcccacatga
      661 ttaagttcag gggccatttc ctgatcgagg gggatctcaa cccggacaat agcgatgttg
      721 acaagctgtt catccagctc gtgcagacgt acaaccagct cttcgaggag aaccccatta
      781 atgcgtcagg cgtcgacgcg aaggctatcc tgtccgctag gctctcgaag tctcggcgcc
      841 tcgagaacct gatcgcccag ctgccgggcg agaagaagaa cggcctgttc gggaatctca
      901 ttgcgctcag cctggggctc acgcccaact tcaagtcgaa tttcgatctc gctgaggacg
      961 ccaagctgca gctctccaag gacacatacg acgatgacct ggataacctc ctggcccaga
     1021 tcggcgatca gtacgcggac ctgttcctcg ctgccaagaa tctgtcggac gccatcctcc
     1081 tgtctgatat tctcagggtg aacaccgaga ttacgaaggc tccgctctca gcctccatga
     1141 tcaagcgcta cgacgagcac catcaggatc tgaccctcct gaaggcgctg gtcaggcagc
     1201 agctccccga gaagtacaag gagatcttct tcgatcagtc gaagaacggc tacgctgggt
     1261 acattgacgg cggggcctct caggaggagt tctacaagtt catcaagccg attctggaga
     1321 agatggacgg cacggaggag ctgctggtga agctcaatcg cgaggacctc ctgaggaagc
     1381 agcggacatt cgataacggc agcatcccac accagattca tctcggggag ctgcacgcta
     1441 tcctgaggag gcaggaggac ttctaccctt tcctcaagga taaccgcgag aagatcgaga
     1501 agattctgac tttcaggatc ccgtactacg tcggcccact cgctaggggc aactcccgct
     1561 tcgcttggat gacccgcaag tcagaggaga cgatcacgcc gtggaacttc gaggaggtgg
     1621 tcgacaaggg cgctagcgct cagtcgttca tcgagaggat gacgaatttc gacaagaacc
     1681 tgccaaatga gaaggtgctc cctaagcact cgctcctgta cgagtacttc acagtctaca
     1741 acgagctgac taaggtgaag tatgtgaccg agggcatgag gaagccggct ttcctgtctg
     1801 gggagcagaa gaaggccatc gtggacctcc tgttcaagac caaccggaag gtcacggtta
     1861 agcagctcaa ggaggactac ttcaagaaga ttgagtgctt cgattcggtc gagatctctg
     1921 gcgttgagga ccgcttcaac gcctccctgg ggacctacca cgatctcctg aagatcatta
     1981 aggataagga cttcctggac aacgaggaga atgaggatat cctcgaggac attgtgctga
     2041 cactcactct gttcgaggac cgggagatga tcgaggagcg cctgaagact tacgcccatc
     2101 tcttcgatga caaggtcatg aagcagctca agaggaggag gtacaccggc tgggggaggc
     2161 tgagcaggaa gctcatcaac ggcattcggg acaagcagtc cgggaagacg atcctcgact
     2221 tcctgaagag cgatggcttc gcgaaccgca atttcatgca gctgattcac gatgacagcc
     2281 tcacattcaa ggaggatatc cagaaggctc aggtgagcgg ccagggggac tcgctgcacg
     2341 agcatatcgc gaacctcgct ggctcgccag ctatcaagaa ggggattctg cagaccgtga
     2401 aggttgtgga cgagctggtg aaggtcatgg gcaggcacaa gcctgagaac atcgtcattg
     2461 agatggcccg ggagaatcag accacgcaga agggccagaa gaactcacgc gagaggatga
     2521 agaggatcga ggagggcatt aaggagctgg ggtcccagat cctcaaggag cacccggtgg
     2581 agaacacgca gctgcagaat gagaagctct acctgtacta cctccagaat ggccgcgata
     2641 tgtatgtgga ccaggagctg gatattaaca ggctcagcga ttacgacgtc gatcatatcg
     2701 ttccacagtc attcctgaag gatgactcca ttgacaacaa ggtcctcacc aggtcggaca
     2761 agaaccgggg caagtctgat aatgttcctt cagaggaggt cgttaagaag atgaagaact
     2821 actggcgcca gctcctgaat gccaagctga tcacgcagcg gaagttcgat aacctcacaa
     2881 aggctgagag gggcgggctc tctgagctgg acaaggcggg cttcatcaag aggcagctgg
     2941 tcgagacacg gcagatcact aagcacgttg cgcagattct cgactcacgg atgaacacta
     3001 agtacgatga gaatgacaag ctgatccgcg aggtgaaggt catcaccctg aagtcaaagc
     3061 tcgtctccga cttcaggaag gatttccagt tctacaaggt tcgggagatc aacaattacc
     3121 accatgccca tgacgcgtac ctgaacgcgg tggtcggcac agctctgatc aagaagtacc
     3181 caaagctcga gagcgagttc gtgtacgggg actacaaggt ttacgatgtg aggaagatga
     3241 tcgccaagtc ggagcaggag attggcaagg ctaccgccaa gtacttcttc tactctaaca
     3301 ttatgaattt cttcaagaca gagatcactc tggccaatgg cgagatccgg aagcgccccc
     3361 tcatcgagac gaacggcgag acgggggaga tcgtgtggga caagggcagg gatttcgcga
     3421 ccgtcaggaa ggttctctcc atgccacaag tgaatatcgt caagaagaca gaggtccaga
     3481 ctggcgggtt ctctaaggag tcaattctgc ctaagcggaa cagcgacaag ctcatcgccc
     3541 gcaagaagga ctgggatccg aagaagtacg gcgggttcga cagccccact gtggcctact
     3601 cggtcctggt tgtggcgaag gttgagaagg gcaagtccaa gaagctcaag agcgtgaagg
     3661 agctgctggg gatcacgatt atggagcgct ccagcttcga gaagaacccg atcgatttcc
     3721 tggaggcgaa gggctacaag gaggtgaaga aggacctgat cattaagctc cccaagtact
     3781 cactcttcga gctggagaac ggcaggaagc ggatgctggc ttccgctggc gagctgcaga
     3841 aggggaacga gctggctctg ccgtccaagt atgtgaactt cctctacctg gcctcccact
     3901 acgagaagct caagggcagc cccgaggaca acgagcagaa gcagctgttc gtcgagcagc
     3961 acaagcatta cctcgacgag atcattgagc agatttccga gttctccaag cgcgtgatcc
     4021 tggccgacgc gaatctggat aaggtcctct ccgcgtacaa caagcaccgc gacaagccaa
     4081 tcagggagca ggctgagaat atcattcatc tcttcaccct gacgaacctc ggcgcccctg
     4141 ctgctttcaa gtacttcgac acaactatcg atcgcaagag gtacacaagc actaaggagg
     4201 tcctggacgc gaccctcatc caccagtcga ttaccggcct ctacgagacg cgcatcgacc
     4261 tgtctcagct cgggggcgac aagcggccag cggcgacgaa gaaggcgggg caggcgaaga
     4321 agaagaagtg aactagtgtc atgggtcgtt taagctgccg atgtgcctgc gtcgtctggt
     4381 gccctctctc catatggagg ttgtcaaagt atctgctgtt cgtgtcatga gtcgtgtcag
     4441 tgttggttta ataatggacc ggttgtgttg tgtgtgcgta ctacccagaa ctatgacaaa
     4501 tcatgaataa gtttgatgtt tgaaattaaa gcctgtgctc attatgttct gtctttcagt
     4561 tgtctcctaa tatttgcctg cagaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4621 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4681 aaaaaaaaaa aaaaaaaaaa aaagagacct ccctcacacc ggtgacgggg atcgcatgcg
     4741 atatctcgag atctagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4801 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4861 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4921 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4981 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5041 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5101 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5161 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5221 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5281 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5341 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5401 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5461 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5521 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5581 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5641 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5701 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5761 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5821 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5881 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5941 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6001 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6061 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6121 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6181 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6241 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6301 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6361 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6421 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6481 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6541 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6601 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6661 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6721 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6781 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6841 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6901 catttccccg aaaagtgcca cctg
//
