LOCUS       Exported                7499 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 7499)
  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..7499
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     12..65
                     /label=T7-TMV-Cas9-F
     primer_bind     12..65
                     /label=T7-TMV-BE-F
     promoter        13..32
                     /label=T7
     misc_feature    33..100
                     /label=TMV
     primer_bind     complement(68..121)
                     /label=TMV-ABE-R
     primer_bind     100..138
                     /label=T7-ABE8e-F
     misc_feature    101..601
                     /label=ABE8
     primer_bind     551..574
                     /label=CX-ABE8-F
     primer_bind     complement(553..571)
                     /label=JC-ABE8-R
     misc_feature    602..697
                     /label=32aalinker
     primer_bind     complement(672..722)
                     /label=wtTadDEL7.15-Gib-R
     primer_bind     complement(694..721)
                     /label=PH-ABE8-R
     CDS             704..4804
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     1177..1196
                     /label=pKSE411-2F
     primer_bind     1833..1852
                     /label=pKSE411-3F
     primer_bind     2553..2572
                     /label=pKSE411-4F
     primer_bind     3249..3268
                     /label=pKSE411-5F
     primer_bind     3968..3987
                     /label=pKSE411-6F
     primer_bind     4681..4700
                     /label=pKSE411-7F
     CDS             4817..4837
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     4817..4835
                     /label=T7-ZmCas9-F
     CDS             4850..4870
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4879..4931)
                     /label=T7-ABE8e-R
     CDS             4883..4903
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4907
                     /label=E9ter
     misc_feature    4913..5158
                     /label=M-ZmUBI-3UTR
     misc_feature    5279..5284
                     /label=BsaI
     primer_bind     complement(5347..5363)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5371..5387
                     /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(5395..5425)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5440..5461
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5749..6337)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6508..7368)
                     /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     7332..7351
                     /label=CX-Amp-F
     promoter        complement(7369..7473)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagctcggta cctaatacga ctcactatag gggtattttt acaacaatta ccaacaacaa
       61 caaacaacaa acaacattac aattactatt tacaattaca atgagcgaag ttgaatttag
      121 ccatgaatat tggatgcgcc atgcacttac acttgcaaag agagcccgcg atgaacgcga
      181 agttcctgtt ggagcagttc ttgttcttaa caaccgcgtt atcggagaag gatggaaccg
      241 cgcaatcgga cttcatgatc ctacagcaca tgcagaaatc atggcacttc gccagggagg
      301 acttgttatg cagaactatc gccttatcga tgcaacactt tatgttacat ttgaaccttg
      361 cgttatgtgc gcaggagcaa tgatccatag ccgcatcgga cgcgttgtgt tcggagtacg
      421 taacagcaaa cgcggagcag caggaagcct tatgaacgtt cttaactatc ctggaatgaa
      481 ccatcgcgtt gaaatcacag aaggaatcct tgcagatgaa tgcgcagcac ttctttgcga
      541 tttctaccgt atgcctcgcc aggtgttcaa tgcgcagaag aaggcccaaa gcagcatcaa
      601 ctcaggtggt agctccggcg gctcgtcagg ttccgaaaca cctggaacaa gcgaaagcgc
      661 aacacctgaa tctagtggag gaagttctgg tgggtccctt aaggacaaga agtactcgat
      721 cggcctcgcc attgggacta actctgttgg ctgggccgtg atcaccgacg agtacaaggt
      781 gccctcaaag aagttcaagg tcctgggcaa caccgatcgg cattccatca agaagaatct
      841 cattggcgct ctcctgttcg acagcggcga gacggctgag gctacgcggc tcaagcgcac
      901 cgcccgcagg cggtacacgc gcaggaagaa tcgcatctgc tacctgcagg agattttctc
      961 caacgagatg gcgaaggttg acgattcttt cttccacagg ctggaggagt cattcctcgt
     1021 ggaggaggat aagaagcacg agcggcatcc aatcttcggc aacattgtcg acgaggttgc
     1081 ctaccacgag aagtacccta cgatctacca tctgcggaag aagctcgtgg actccacaga
     1141 taaggcggac ctccgcctga tctacctcgc tctggcccac atgattaagt tcaggggcca
     1201 tttcctgatc gagggggatc tcaacccgga caatagcgat gttgacaagc tgttcatcca
     1261 gctcgtgcag acgtacaacc agctcttcga ggagaacccc attaatgcgt caggcgtcga
     1321 cgcgaaggct atcctgtccg ctaggctctc gaagtctcgg cgcctcgaga acctgatcgc
     1381 ccagctgccg ggcgagaaga agaacggcct gttcgggaat ctcattgcgc tcagcctggg
     1441 gctcacgccc aacttcaagt cgaatttcga tctcgctgag gacgccaagc tgcagctctc
     1501 caaggacaca tacgacgatg acctggataa cctcctggcc cagatcggcg atcagtacgc
     1561 ggacctgttc ctcgctgcca agaatctgtc ggacgccatc ctcctgtctg atattctcag
     1621 ggtgaacacc gagattacga aggctccgct ctcagcctcc atgatcaagc gctacgacga
     1681 gcaccatcag gatctgaccc tcctgaaggc gctggtcagg cagcagctcc ccgagaagta
     1741 caaggagatc ttcttcgatc agtcgaagaa cggctacgct gggtacattg acggcggggc
     1801 ctctcaggag gagttctaca agttcatcaa gccgattctg gagaagatgg acggcacgga
     1861 ggagctgctg gtgaagctca atcgcgagga cctcctgagg aagcagcgga cattcgataa
     1921 cggcagcatc ccacaccaga ttcatctcgg ggagctgcac gctatcctga ggaggcagga
     1981 ggacttctac cctttcctca aggataaccg cgagaagatc gagaagattc tgactttcag
     2041 gatcccgtac tacgtcggcc cactcgctag gggcaactcc cgcttcgctt ggatgacccg
     2101 caagtcagag gagacgatca cgccgtggaa cttcgaggag gtggtcgaca agggcgctag
     2161 cgctcagtcg ttcatcgaga ggatgacgaa tttcgacaag aacctgccaa atgagaaggt
     2221 gctccctaag cactcgctcc tgtacgagta cttcacagtc tacaacgagc tgactaaggt
     2281 gaagtatgtg accgagggca tgaggaagcc ggctttcctg tctggggagc agaagaaggc
     2341 catcgtggac ctcctgttca agaccaaccg gaaggtcacg gttaagcagc tcaaggagga
     2401 ctacttcaag aagattgagt gcttcgattc ggtcgagatc tctggcgttg aggaccgctt
     2461 caacgcctcc ctggggacct accacgatct cctgaagatc attaaggata aggacttcct
     2521 ggacaacgag gagaatgagg atatcctcga ggacattgtg ctgacactca ctctgttcga
     2581 ggaccgggag atgatcgagg agcgcctgaa gacttacgcc catctcttcg atgacaaggt
     2641 catgaagcag ctcaagagga ggaggtacac cggctggggg aggctgagca ggaagctcat
     2701 caacggcatt cgggacaagc agtccgggaa gacgatcctc gacttcctga agagcgatgg
     2761 cttcgcgaac cgcaatttca tgcagctgat tcacgatgac agcctcacat tcaaggagga
     2821 tatccagaag gctcaggtga gcggccaggg ggactcgctg cacgagcata tcgcgaacct
     2881 cgctggctcg ccagctatca agaaggggat tctgcagacc gtgaaggttg tggacgagct
     2941 ggtgaaggtc atgggcaggc acaagcctga gaacatcgtc attgagatgg cccgggagaa
     3001 tcagaccacg cagaagggcc agaagaactc acgcgagagg atgaagagga tcgaggaggg
     3061 cattaaggag ctggggtccc agatcctcaa ggagcacccg gtggagaaca cgcagctgca
     3121 gaatgagaag ctctacctgt actacctcca gaatggccgc gatatgtatg tggaccagga
     3181 gctggatatt aacaggctca gcgattacga cgtcgatcat atcgttccac agtcattcct
     3241 gaaggatgac tccattgaca acaaggtcct caccaggtcg gacaagaacc ggggcaagtc
     3301 tgataatgtt ccttcagagg aggtcgttaa gaagatgaag aactactggc gccagctcct
     3361 gaatgccaag ctgatcacgc agcggaagtt cgataacctc acaaaggctg agaggggcgg
     3421 gctctctgag ctggacaagg cgggcttcat caagaggcag ctggtcgaga cacggcagat
     3481 cactaagcac gttgcgcaga ttctcgactc acggatgaac actaagtacg atgagaatga
     3541 caagctgatc cgcgaggtga aggtcatcac cctgaagtca aagctcgtct ccgacttcag
     3601 gaaggatttc cagttctaca aggttcggga gatcaacaat taccaccatg cccatgacgc
     3661 gtacctgaac gcggtggtcg gcacagctct gatcaagaag tacccaaagc tcgagagcga
     3721 gttcgtgtac ggggactaca aggtttacga tgtgaggaag atgatcgcca agtcggagca
     3781 ggagattggc aaggctaccg ccaagtactt cttctactct aacattatga atttcttcaa
     3841 gacagagatc actctggcca atggcgagat ccggaagcgc cccctcatcg agacgaacgg
     3901 cgagacgggg gagatcgtgt gggacaaggg cagggatttc gcgaccgtca ggaaggttct
     3961 ctccatgcca caagtgaata tcgtcaagaa gacagaggtc cagactggcg ggttctctaa
     4021 ggagtcaatt ctgcctaagc ggaacagcga caagctcatc gcccgcaaga aggactggga
     4081 tccgaagaag tacggcgggt tcgacagccc cactgtggcc tactcggtcc tggttgtggc
     4141 gaaggttgag aagggcaagt ccaagaagct caagagcgtg aaggagctgc tggggatcac
     4201 gattatggag cgctccagct tcgagaagaa cccgatcgat ttcctggagg cgaagggcta
     4261 caaggaggtg aagaaggacc tgatcattaa gctccccaag tactcactct tcgagctgga
     4321 gaacggcagg aagcggatgc tggcttccgc tggcgagctg cagaagggga acgagctggc
     4381 tctgccgtcc aagtatgtga acttcctcta cctggcctcc cactacgaga agctcaaggg
     4441 cagccccgag gacaacgagc agaagcagct gttcgtcgag cagcacaagc attacctcga
     4501 cgagatcatt gagcagattt ccgagttctc caagcgcgtg atcctggccg acgcgaatct
     4561 ggataaggtc ctctccgcgt acaacaagca ccgcgacaag ccaatcaggg agcaggctga
     4621 gaatatcatt catctcttca ccctgacgaa cctcggcgcc cctgctgctt tcaagtactt
     4681 cgacacaact atcgatcgca agaggtacac aagcactaag gaggtcctgg acgcgaccct
     4741 catccaccag tcgattaccg gcctctacga gacgcgcatc gacctgtctc agctcggggg
     4801 cgactccggc ggcagcccaa agaagaagcg gaaggtgtct ggaggttctc ctaagaaaaa
     4861 gagaaaagtg tccggcggct ccccgaagaa gaagcgcaag gtgtagacta gtgtcatggg
     4921 tcgtttaagc tgccgatgtg cctgcgtcgt ctggtgccct ctctccatat ggaggttgtc
     4981 aaagtatctg ctgttcgtgt catgagtcgt gtcagtgttg gtttaataat ggaccggttg
     5041 tgttgtgtgt gcgtactacc cagaactatg acaaatcatg aataagtttg atgtttgaaa
     5101 ttaaagcctg tgctcattat gttctgtctt tcagttgtct cctaatattt gcctgcagaa
     5161 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5221 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaga
     5281 gacctccctc acaccggtga cggggatcgc atgcgatatc tcgagatcta gcttggcgta
     5341 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5401 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5461 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5521 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5581 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5641 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5701 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5761 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5821 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5881 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5941 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6001 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6061 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6121 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6181 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6241 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6301 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6361 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6421 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6481 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6541 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6601 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6661 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6721 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6781 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6841 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6901 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6961 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7021 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7081 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7141 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7201 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7261 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7321 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7381 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7441 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
