LOCUS       Exported                6896 bp ds-DNA     circular SYN 02-MAR-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 6896)
  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..6896
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     12..65
                     /label=TMV-XB-F
     primer_bind     12..65
                     /label=T7-TMV-F
     primer_bind     12..65
                     /label=T7-TMV-BE-F
     primer_bind     12..65
                     /label=T7-TMV-Cas9-F
     promoter        13..32
                     /label=T7
     primer_bind     15..65
                     /label=TMV-PE-F
     primer_bind     17..65
                     /label=XB-TMV-F
     misc_feature    33..100
                     /label=TMV
     primer_bind     33..65
                     /label=UBI-TMV-F
     primer_bind     complement(68..120)
                     /label=XB-TMV-R
     primer_bind     complement(68..120)
                     /label=T7-TMV-Cas9-R
     primer_bind     complement(68..116)
                     /label=TMV-XB-R
     primer_bind     complement(68..104)
                     /label=TMV-A3A-R
     primer_bind     complement(68..103)
                     /label=TMV-ABE-R
     primer_bind     complement(68..100)
                     /label=UBI-TMV-R
     primer_bind     complement(68..100)
                     /label=TMV-PE-R
     primer_bind     100..125
                     /label=T7-ZmCas9-F
     CDS             101..4303
                     /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"
     primer_bind     152..173
                     /label=1562-ZMcas9-F
     primer_bind     complement(4280..4300)
                     /label=1562-ZMcas9-R
     primer_bind     complement(4283..4303)
                     /label=T7-ZmCas9-R
     terminator      4304
                     /label=E9ter
     misc_feature    4310..4555
                     /label=M-ZmUBI-3UTR
     misc_feature    4676..4681
                     /label=BsaI
     primer_bind     complement(4744..4760)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4768..4784
                     /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(4792..4822)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4837..4858
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5146..5734)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5905..6765)
                     /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     6729..6748
                     /label=CX-Amp-F
     promoter        complement(6766..6870)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagctcggta cctaatacga ctcactatag gggtattttt acaacaatta ccaacaacaa
       61 caaacaacaa acaacattac aattactatt tacaattaca atggctccga agaagaagag
      121 gaaggttggc atccacgggg tgccagctgc tgacaagaag tactcgatcg gcctcgatat
      181 tgggactaac tctgttggct gggccgtgat caccgacgag tacaaggtgc cctcaaagaa
      241 gttcaaggtc ctgggcaaca ccgatcggca ttccatcaag aagaatctca ttggcgctct
      301 cctgttcgac agcggcgaga cggctgaggc tacgcggctc aagcgcaccg cccgcaggcg
      361 gtacacgcgc aggaagaatc gcatctgcta cctgcaggag attttctcca acgagatggc
      421 gaaggttgac gattctttct tccacaggct ggaggagtca ttcctcgtgg aggaggataa
      481 gaagcacgag cggcatccaa tcttcggcaa cattgtcgac gaggttgcct accacgagaa
      541 gtaccctacg atctaccatc tgcggaagaa gctcgtggac tccacagata aggcggacct
      601 ccgcctgatc tacctcgctc tggcccacat gattaagttc aggggccatt tcctgatcga
      661 gggggatctc aacccggaca atagcgatgt tgacaagctg ttcatccagc tcgtgcagac
      721 gtacaaccag ctcttcgagg agaaccccat taatgcgtca ggcgtcgacg cgaaggctat
      781 cctgtccgct aggctctcga agtctcggcg cctcgagaac ctgatcgccc agctgccggg
      841 cgagaagaag aacggcctgt tcgggaatct cattgcgctc agcctggggc tcacgcccaa
      901 cttcaagtcg aatttcgatc tcgctgagga cgccaagctg cagctctcca aggacacata
      961 cgacgatgac ctggataacc tcctggccca gatcggcgat cagtacgcgg acctgttcct
     1021 cgctgccaag aatctgtcgg acgccatcct cctgtctgat attctcaggg tgaacaccga
     1081 gattacgaag gctccgctct cagcctccat gatcaagcgc tacgacgagc accatcagga
     1141 tctgaccctc ctgaaggcgc tggtcaggca gcagctcccc gagaagtaca aggagatctt
     1201 cttcgatcag tcgaagaacg gctacgctgg gtacattgac ggcggggcct ctcaggagga
     1261 gttctacaag ttcatcaagc cgattctgga gaagatggac ggcacggagg agctgctggt
     1321 gaagctcaat cgcgaggacc tcctgaggaa gcagcggaca ttcgataacg gcagcatccc
     1381 acaccagatt catctcgggg agctgcacgc tatcctgagg aggcaggagg acttctaccc
     1441 tttcctcaag gataaccgcg agaagatcga gaagattctg actttcagga tcccgtacta
     1501 cgtcggccca ctcgctaggg gcaactcccg cttcgcttgg atgacccgca agtcagagga
     1561 gacgatcacg ccgtggaact tcgaggaggt ggtcgacaag ggcgctagcg ctcagtcgtt
     1621 catcgagagg atgacgaatt tcgacaagaa cctgccaaat gagaaggtgc tccctaagca
     1681 ctcgctcctg tacgagtact tcacagtcta caacgagctg actaaggtga agtatgtgac
     1741 cgagggcatg aggaagccgg ctttcctgtc tggggagcag aagaaggcca tcgtggacct
     1801 cctgttcaag accaaccgga aggtcacggt taagcagctc aaggaggact acttcaagaa
     1861 gattgagtgc ttcgattcgg tcgagatctc tggcgttgag gaccgcttca acgcctccct
     1921 ggggacctac cacgatctcc tgaagatcat taaggataag gacttcctgg acaacgagga
     1981 gaatgaggat atcctcgagg acattgtgct gacactcact ctgttcgagg accgggagat
     2041 gatcgaggag cgcctgaaga cttacgccca tctcttcgat gacaaggtca tgaagcagct
     2101 caagaggagg aggtacaccg gctgggggag gctgagcagg aagctcatca acggcattcg
     2161 ggacaagcag tccgggaaga cgatcctcga cttcctgaag agcgatggct tcgcgaaccg
     2221 caatttcatg cagctgattc acgatgacag cctcacattc aaggaggata tccagaaggc
     2281 tcaggtgagc ggccaggggg actcgctgca cgagcatatc gcgaacctcg ctggctcgcc
     2341 agctatcaag aaggggattc tgcagaccgt gaaggttgtg gacgagctgg tgaaggtcat
     2401 gggcaggcac aagcctgaga acatcgtcat tgagatggcc cgggagaatc agaccacgca
     2461 gaagggccag aagaactcac gcgagaggat gaagaggatc gaggagggca ttaaggagct
     2521 ggggtcccag atcctcaagg agcacccggt ggagaacacg cagctgcaga atgagaagct
     2581 ctacctgtac tacctccaga atggccgcga tatgtatgtg gaccaggagc tggatattaa
     2641 caggctcagc gattacgacg tcgatcatat cgttccacag tcattcctga aggatgactc
     2701 cattgacaac aaggtcctca ccaggtcgga caagaaccgg ggcaagtctg ataatgttcc
     2761 ttcagaggag gtcgttaaga agatgaagaa ctactggcgc cagctcctga atgccaagct
     2821 gatcacgcag cggaagttcg ataacctcac aaaggctgag aggggcgggc tctctgagct
     2881 ggacaaggcg ggcttcatca agaggcagct ggtcgagaca cggcagatca ctaagcacgt
     2941 tgcgcagatt ctcgactcac ggatgaacac taagtacgat gagaatgaca agctgatccg
     3001 cgaggtgaag gtcatcaccc tgaagtcaaa gctcgtctcc gacttcagga aggatttcca
     3061 gttctacaag gttcgggaga tcaacaatta ccaccatgcc catgacgcgt acctgaacgc
     3121 ggtggtcggc acagctctga tcaagaagta cccaaagctc gagagcgagt tcgtgtacgg
     3181 ggactacaag gtttacgatg tgaggaagat gatcgccaag tcggagcagg agattggcaa
     3241 ggctaccgcc aagtacttct tctactctaa cattatgaat ttcttcaaga cagagatcac
     3301 tctggccaat ggcgagatcc ggaagcgccc cctcatcgag acgaacggcg agacggggga
     3361 gatcgtgtgg gacaagggca gggatttcgc gaccgtcagg aaggttctct ccatgccaca
     3421 agtgaatatc gtcaagaaga cagaggtcca gactggcggg ttctctaagg agtcaattct
     3481 gcctaagcgg aacagcgaca agctcatcgc ccgcaagaag gactgggatc cgaagaagta
     3541 cggcgggttc gacagcccca ctgtggccta ctcggtcctg gttgtggcga aggttgagaa
     3601 gggcaagtcc aagaagctca agagcgtgaa ggagctgctg gggatcacga ttatggagcg
     3661 ctccagcttc gagaagaacc cgatcgattt cctggaggcg aagggctaca aggaggtgaa
     3721 gaaggacctg atcattaagc tccccaagta ctcactcttc gagctggaga acggcaggaa
     3781 gcggatgctg gcttccgctg gcgagctgca gaaggggaac gagctggctc tgccgtccaa
     3841 gtatgtgaac ttcctctacc tggcctccca ctacgagaag ctcaagggca gccccgagga
     3901 caacgagcag aagcagctgt tcgtcgagca gcacaagcat tacctcgacg agatcattga
     3961 gcagatttcc gagttctcca agcgcgtgat cctggccgac gcgaatctgg ataaggtcct
     4021 ctccgcgtac aacaagcacc gcgacaagcc aatcagggag caggctgaga atatcattca
     4081 tctcttcacc ctgacgaacc tcggcgcccc tgctgctttc aagtacttcg acacaactat
     4141 cgatcgcaag aggtacacaa gcactaagga ggtcctggac gcgaccctca tccaccagtc
     4201 gattaccggc ctctacgaga cgcgcatcga cctgtctcag ctcgggggcg acaagcggcc
     4261 agcggcgacg aagaaggcgg ggcaggcgaa gaagaagaag tgaactagtg tcatgggtcg
     4321 tttaagctgc cgatgtgcct gcgtcgtctg gtgccctctc tccatatgga ggttgtcaaa
     4381 gtatctgctg ttcgtgtcat gagtcgtgtc agtgttggtt taataatgga ccggttgtgt
     4441 tgtgtgtgcg tactacccag aactatgaca aatcatgaat aagtttgatg tttgaaatta
     4501 aagcctgtgc tcattatgtt ctgtctttca gttgtctcct aatatttgcc tgcagaaaaa
     4561 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4621 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaagagac
     4681 ctccctcaca ccggtgacgg ggatcgcatg cgatatctcg agatctagct tggcgtaatc
     4741 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4801 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4861 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4921 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4981 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5041 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5101 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5161 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5221 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5281 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5341 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5401 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5461 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5521 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5581 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5641 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5701 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5761 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5821 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5881 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5941 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6001 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6061 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6121 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6181 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6241 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6301 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6361 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6421 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6481 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6541 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6601 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6661 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6721 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6781 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6841 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctg
//
