LOCUS       Exported                7527 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 7527)
  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..7527
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     13..54
                     /label=DEN-ABE-F
     promoter        13..32
                     /label=T7
     misc_feature    33..128
                     /label=DEN2
     primer_bind     complement(100..146)
                     /label=DEN-ABE-R
     misc_feature    129..629
                     /label=ABE8
     primer_bind     579..602
                     /label=CX-ABE8-F
     primer_bind     complement(581..599)
                     /label=JC-ABE8-R
     misc_feature    630..725
                     /label=32aalinker
     primer_bind     complement(700..750)
                     /label=wtTadDEL7.15-Gib-R
     primer_bind     complement(722..749)
                     /label=PH-ABE8-R
     CDS             732..4832
                     /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     1205..1224
                     /label=pKSE411-2F
     primer_bind     1861..1880
                     /label=pKSE411-3F
     primer_bind     2581..2600
                     /label=pKSE411-4F
     primer_bind     3277..3296
                     /label=pKSE411-5F
     primer_bind     3996..4015
                     /label=pKSE411-6F
     primer_bind     4709..4728
                     /label=pKSE411-7F
     CDS             4845..4865
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4878..4898
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4907..4959)
                     /label=T7-ABE8e-R
     CDS             4911..4931
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4935
                     /label=E9ter
     misc_feature    4941..5186
                     /label=M-ZmUBI-3UTR
     misc_feature    5307..5312
                     /label=BsaI
     primer_bind     complement(5375..5391)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5399..5415
                     /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(5423..5453)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5468..5489
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5777..6365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6536..7396)
                     /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     7360..7379
                     /label=CX-Amp-F
     promoter        complement(7397..7501)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagctcggta cctaatacga ctcactatag ggagttgtta gtctacgtgg accgacaaag
       61 acagattctt tgagggagct aagctcaacg tagttctaac agttttttaa ttagagagca
      121 gatctctgat gagcgaagtt gaatttagcc atgaatattg gatgcgccat gcacttacac
      181 ttgcaaagag agcccgcgat gaacgcgaag ttcctgttgg agcagttctt gttcttaaca
      241 accgcgttat cggagaagga tggaaccgcg caatcggact tcatgatcct acagcacatg
      301 cagaaatcat ggcacttcgc cagggaggac ttgttatgca gaactatcgc cttatcgatg
      361 caacacttta tgttacattt gaaccttgcg ttatgtgcgc aggagcaatg atccatagcc
      421 gcatcggacg cgttgtgttc ggagtacgta acagcaaacg cggagcagca ggaagcctta
      481 tgaacgttct taactatcct ggaatgaacc atcgcgttga aatcacagaa ggaatccttg
      541 cagatgaatg cgcagcactt ctttgcgatt tctaccgtat gcctcgccag gtgttcaatg
      601 cgcagaagaa ggcccaaagc agcatcaact caggtggtag ctccggcggc tcgtcaggtt
      661 ccgaaacacc tggaacaagc gaaagcgcaa cacctgaatc tagtggagga agttctggtg
      721 ggtcccttaa ggacaagaag tactcgatcg gcctcgccat tgggactaac tctgttggct
      781 gggccgtgat caccgacgag tacaaggtgc cctcaaagaa gttcaaggtc ctgggcaaca
      841 ccgatcggca ttccatcaag aagaatctca ttggcgctct cctgttcgac agcggcgaga
      901 cggctgaggc tacgcggctc aagcgcaccg cccgcaggcg gtacacgcgc aggaagaatc
      961 gcatctgcta cctgcaggag attttctcca acgagatggc gaaggttgac gattctttct
     1021 tccacaggct ggaggagtca ttcctcgtgg aggaggataa gaagcacgag cggcatccaa
     1081 tcttcggcaa cattgtcgac gaggttgcct accacgagaa gtaccctacg atctaccatc
     1141 tgcggaagaa gctcgtggac tccacagata aggcggacct ccgcctgatc tacctcgctc
     1201 tggcccacat gattaagttc aggggccatt tcctgatcga gggggatctc aacccggaca
     1261 atagcgatgt tgacaagctg ttcatccagc tcgtgcagac gtacaaccag ctcttcgagg
     1321 agaaccccat taatgcgtca ggcgtcgacg cgaaggctat cctgtccgct aggctctcga
     1381 agtctcggcg cctcgagaac ctgatcgccc agctgccggg cgagaagaag aacggcctgt
     1441 tcgggaatct cattgcgctc agcctggggc tcacgcccaa cttcaagtcg aatttcgatc
     1501 tcgctgagga cgccaagctg cagctctcca aggacacata cgacgatgac ctggataacc
     1561 tcctggccca gatcggcgat cagtacgcgg acctgttcct cgctgccaag aatctgtcgg
     1621 acgccatcct cctgtctgat attctcaggg tgaacaccga gattacgaag gctccgctct
     1681 cagcctccat gatcaagcgc tacgacgagc accatcagga tctgaccctc ctgaaggcgc
     1741 tggtcaggca gcagctcccc gagaagtaca aggagatctt cttcgatcag tcgaagaacg
     1801 gctacgctgg gtacattgac ggcggggcct ctcaggagga gttctacaag ttcatcaagc
     1861 cgattctgga gaagatggac ggcacggagg agctgctggt gaagctcaat cgcgaggacc
     1921 tcctgaggaa gcagcggaca ttcgataacg gcagcatccc acaccagatt catctcgggg
     1981 agctgcacgc tatcctgagg aggcaggagg acttctaccc tttcctcaag gataaccgcg
     2041 agaagatcga gaagattctg actttcagga tcccgtacta cgtcggccca ctcgctaggg
     2101 gcaactcccg cttcgcttgg atgacccgca agtcagagga gacgatcacg ccgtggaact
     2161 tcgaggaggt ggtcgacaag ggcgctagcg ctcagtcgtt catcgagagg atgacgaatt
     2221 tcgacaagaa cctgccaaat gagaaggtgc tccctaagca ctcgctcctg tacgagtact
     2281 tcacagtcta caacgagctg actaaggtga agtatgtgac cgagggcatg aggaagccgg
     2341 ctttcctgtc tggggagcag aagaaggcca tcgtggacct cctgttcaag accaaccgga
     2401 aggtcacggt taagcagctc aaggaggact acttcaagaa gattgagtgc ttcgattcgg
     2461 tcgagatctc tggcgttgag gaccgcttca acgcctccct ggggacctac cacgatctcc
     2521 tgaagatcat taaggataag gacttcctgg acaacgagga gaatgaggat atcctcgagg
     2581 acattgtgct gacactcact ctgttcgagg accgggagat gatcgaggag cgcctgaaga
     2641 cttacgccca tctcttcgat gacaaggtca tgaagcagct caagaggagg aggtacaccg
     2701 gctgggggag gctgagcagg aagctcatca acggcattcg ggacaagcag tccgggaaga
     2761 cgatcctcga cttcctgaag agcgatggct tcgcgaaccg caatttcatg cagctgattc
     2821 acgatgacag cctcacattc aaggaggata tccagaaggc tcaggtgagc ggccaggggg
     2881 actcgctgca cgagcatatc gcgaacctcg ctggctcgcc agctatcaag aaggggattc
     2941 tgcagaccgt gaaggttgtg gacgagctgg tgaaggtcat gggcaggcac aagcctgaga
     3001 acatcgtcat tgagatggcc cgggagaatc agaccacgca gaagggccag aagaactcac
     3061 gcgagaggat gaagaggatc gaggagggca ttaaggagct ggggtcccag atcctcaagg
     3121 agcacccggt ggagaacacg cagctgcaga atgagaagct ctacctgtac tacctccaga
     3181 atggccgcga tatgtatgtg gaccaggagc tggatattaa caggctcagc gattacgacg
     3241 tcgatcatat cgttccacag tcattcctga aggatgactc cattgacaac aaggtcctca
     3301 ccaggtcgga caagaaccgg ggcaagtctg ataatgttcc ttcagaggag gtcgttaaga
     3361 agatgaagaa ctactggcgc cagctcctga atgccaagct gatcacgcag cggaagttcg
     3421 ataacctcac aaaggctgag aggggcgggc tctctgagct ggacaaggcg ggcttcatca
     3481 agaggcagct ggtcgagaca cggcagatca ctaagcacgt tgcgcagatt ctcgactcac
     3541 ggatgaacac taagtacgat gagaatgaca agctgatccg cgaggtgaag gtcatcaccc
     3601 tgaagtcaaa gctcgtctcc gacttcagga aggatttcca gttctacaag gttcgggaga
     3661 tcaacaatta ccaccatgcc catgacgcgt acctgaacgc ggtggtcggc acagctctga
     3721 tcaagaagta cccaaagctc gagagcgagt tcgtgtacgg ggactacaag gtttacgatg
     3781 tgaggaagat gatcgccaag tcggagcagg agattggcaa ggctaccgcc aagtacttct
     3841 tctactctaa cattatgaat ttcttcaaga cagagatcac tctggccaat ggcgagatcc
     3901 ggaagcgccc cctcatcgag acgaacggcg agacggggga gatcgtgtgg gacaagggca
     3961 gggatttcgc gaccgtcagg aaggttctct ccatgccaca agtgaatatc gtcaagaaga
     4021 cagaggtcca gactggcggg ttctctaagg agtcaattct gcctaagcgg aacagcgaca
     4081 agctcatcgc ccgcaagaag gactgggatc cgaagaagta cggcgggttc gacagcccca
     4141 ctgtggccta ctcggtcctg gttgtggcga aggttgagaa gggcaagtcc aagaagctca
     4201 agagcgtgaa ggagctgctg gggatcacga ttatggagcg ctccagcttc gagaagaacc
     4261 cgatcgattt cctggaggcg aagggctaca aggaggtgaa gaaggacctg atcattaagc
     4321 tccccaagta ctcactcttc gagctggaga acggcaggaa gcggatgctg gcttccgctg
     4381 gcgagctgca gaaggggaac gagctggctc tgccgtccaa gtatgtgaac ttcctctacc
     4441 tggcctccca ctacgagaag ctcaagggca gccccgagga caacgagcag aagcagctgt
     4501 tcgtcgagca gcacaagcat tacctcgacg agatcattga gcagatttcc gagttctcca
     4561 agcgcgtgat cctggccgac gcgaatctgg ataaggtcct ctccgcgtac aacaagcacc
     4621 gcgacaagcc aatcagggag caggctgaga atatcattca tctcttcacc ctgacgaacc
     4681 tcggcgcccc tgctgctttc aagtacttcg acacaactat cgatcgcaag aggtacacaa
     4741 gcactaagga ggtcctggac gcgaccctca tccaccagtc gattaccggc ctctacgaga
     4801 cgcgcatcga cctgtctcag ctcgggggcg actccggcgg cagcccaaag aagaagcgga
     4861 aggtgtctgg aggttctcct aagaaaaaga gaaaagtgtc cggcggctcc ccgaagaaga
     4921 agcgcaaggt gtagactagt gtcatgggtc gtttaagctg ccgatgtgcc tgcgtcgtct
     4981 ggtgccctct ctccatatgg aggttgtcaa agtatctgct gttcgtgtca tgagtcgtgt
     5041 cagtgttggt ttaataatgg accggttgtg ttgtgtgtgc gtactaccca gaactatgac
     5101 aaatcatgaa taagtttgat gtttgaaatt aaagcctgtg ctcattatgt tctgtctttc
     5161 agttgtctcc taatatttgc ctgcagaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5221 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5281 aaaaaaaaaa aaaaaaaaaa aaaaaagaga cctccctcac accggtgacg gggatcgcat
     5341 gcgatatctc gagatctagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5401 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5461 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5521 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5581 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5641 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5701 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5761 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5821 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5881 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5941 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6001 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6061 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6121 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6181 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6241 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6301 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6361 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6421 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6481 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6541 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6601 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6661 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6721 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6781 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6841 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6901 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6961 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7021 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7081 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7141 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7201 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7261 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7321 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7381 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7441 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7501 gcacatttcc ccgaaaagtg ccacctg
//
