LOCUS       sequence-373765-        6955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  prokaryotic expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6955)
  AUTHORS   Chen Y, Hu Y, Wang X, Luo S, Yang N, Chen Y, Li Z, Zhou Q, Li W
  TITLE     Synergistic engineering of CRISPR-Cas nucleases enables robust 
            mammalian genome editing.
  JOURNAL   Innovation (Camb). 2022 May 26;3(4):100264. doi: 
            10.1016/j.xinn.2022.100264. eCollection 2022 Jul 12.
  PUBMED    35693153
REFERENCE   2  (bases 1 to 6955)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Innovation 
            (Camb)."; date: "2022-05-26"; volume: "3(4):100264. doi"; pages: " 
            10.1016/j.xinn.2022.100264. eCollection 2022 Jul 12"
COMMENT     Sequence Label: BPK2014-Cas12i2
FEATURES             Location/Qualifiers
     source          1..6955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    46..70
                     /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)."
     CDS             117..137
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3312..3341
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     promoter        3410..3428
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3450..3497
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3718..4377)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4292..4311
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4378..4480)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4872..4889)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5006..5551)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(5086..5105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5776..6858)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6859..6936)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cttatgcgac tcctgcatta ggaaattaat acgactcact ataggggaat tgtgagcgga
       61 taacaattcc cctgtagaaa taattttgtt taactttaat aaggagatat catatgccca
      121 agaagaagag gaaggttccc gggatgagca gcgccatcaa gagctacaag agcgtgctgc
      181 gccccaacga gcgcaagaac cagctgctga agagcaccat ccagtgcctg gaggacggca
      241 gcgccttctt cttcaagatg ctgcagggcc tgttcggcgg catcaccccc gagatcgtgc
      301 gcttcagcac cgagcaggag aagcagcagc aggacatcgc cctgtggtgc gccgtgaact
      361 ggttccgccc cgtgagccag gacagcctga cccacaccat cgccagcgac aacctggtgg
      421 agaagttcga ggagtactac ggcggcaccg ccagcgacgc catcaagcag tacttcagcg
      481 ccagcatcgg cgagagctac tactggaacg actgccgcca gcagtactac gacctgtgcc
      541 gcgagctggg cgtggaggtg agcgacctga cccacgacct ggagatcctg tgccgcgaga
      601 agtgcctggc cgtggccacc gagagcaacc agaacaacag catcatcagc gtgctgttcg
      661 gcaccggcga gaaggaggac cgcagcgtga agctgcgcat caccaagaag atcctggagg
      721 ccatcagcaa cctgaaggag atccccaaga acgtggcccc catccaggag atcatcctga
      781 acgtggccaa ggccaccaag gagaccttcc gccaggtgta cgccggcaac ctgggcgccc
      841 ccagcaccct ggagaagttc atcgccaagg acggccagaa ggagttcgac ctgaagaagc
      901 tgcagaccga cctgaagaag gtgatccgcg gcaagagcaa ggagcgcgac tggtgctgcc
      961 aggaggagct gcgcagctac gtggagcaga acaccatcca gtacgacctg tgggcctggg
     1021 gcgagatgtt caacaaggcc cacaccgccc tgaagatcaa gagcacccgc aactacaact
     1081 tcgccaagca gcgcctggag cagttcaagg agatccagag cctgaacaac ctgctggtgg
     1141 tgaagaagct gaacgacttc ttcgacagcg agttcttcag cggcgaggag acctacacca
     1201 tctgcgtgca ccacctgggc ggcaaggacc tgagcaagct gtacaaggcc tgggaggacg
     1261 accccgccga ccccgagaac gccatcgtgg tgctgtgcga cgacctgaag aacaacttca
     1321 agaaggagcc catccgcaac atcctgcgct acatcttcac catccgccag gagtgcagcg
     1381 cccaggacat cctggccgcc gccaagtaca accagcagct ggaccgctac aagagccaga
     1441 aggccaaccc cagcgtgctg ggcaaccagg gcttcacctg gaccaacgcc gtgatcctgc
     1501 ccgagaaggc ccagcgcaac gaccgcccca acagcctgga cctgcgcatc tggctgtacc
     1561 tgaagctgcg ccaccccgac ggccgctgga agaagcacca catccccttc tacgacaccc
     1621 gcttcttcca ggagatctac gccgccggca acagccccgt ggacacctgc cagttccgca
     1681 ccccccgctt cggctaccac ctgcccaagc tgaccgacca gaccgccatc cgcgtgaaca
     1741 agaagcacgt gaaggccgcc aagaccgagg cccgcatccg cctggccatc cagcagggca
     1801 ccctgcccgt gagcaacctg aagatcaccg agatcagcgc caccatcaac agcaagggcc
     1861 aggtgcgcat ccccgtgaag ttcgacgtgg gccgccagaa gggcaccctg cagatcggcg
     1921 accgcttctg cggctacgac cagaaccaga ccgccagcca cgcctacagc ctgtgggagg
     1981 tggtgaagga gggccagtac cacaaggagc tgggctgctt cgtgcgcttc atcagcagcg
     2041 gcgacatcgt gagcatcacc gagaaccgcg gcaaccagtt cgaccagctg agctacgagg
     2101 gcctggccta cccccagtac gccgactggc gcaagaaggc cagcaagttc gtgagcctgt
     2161 ggcagatcac caagaagaac aagaagaagg agatcgtgac cgtggaggcc aaggagaagt
     2221 tcgacgccat ctgcaagtac cagccccgcc tgtacaagtt caacaaggag tacgcctacc
     2281 tgctgcgcga catcgtgcgc ggcaagagcc tggtggagct gcagcagatc cgccaggaga
     2341 tcttccgctt catcgagcag gactgcggcg tgacccgcct gggcagcctg agcctgagca
     2401 ccctggagac cgtgaaggcc gtgaagggca tcatctacag ctacttcagc accgccctga
     2461 acgccagcaa gaacaacccc atcagcgacg agcagcgcaa ggagttcgac cccgagctgt
     2521 tcgccctgct ggagaagctg gagctgatcc gcacccgcaa gaagaagcag aaggtggagc
     2581 gcatcgccaa cagcctgatc cagacctgcc tggagaacaa catcaagttc atccgcggcg
     2641 agggcgacct gagcaccacc aacaacgcca ccaagaagaa ggccaacagc cgcagcatgg
     2701 actggctggc ccgcggcgtg ttcaacaaga tccgccagct ggcccccatg cacaacatca
     2761 ccctgttcgg ctgcggcagc ctgtacacca gccaccagga ccccctggtg caccgcaacc
     2821 ccgacaaggc catgaagtgc cgctgggccg ccatccccgt gaaggacatc ggcgactggg
     2881 tgctgcgcaa gctgagccag aacctgcgcg ccaagaacat cggcaccggc gagtactacc
     2941 accagggcgt gaaggagttc ctgagccact acgagctgca ggacctggag gaggagctgc
     3001 tgaagtggcg cagcgaccgc aagagcaaca tcccctgctg ggtgctgcag aaccgcctgg
     3061 ccgagaagct gggcaacaag gaggccgtgg tgtacatccc cgtgcgcggc ggccgcatct
     3121 acttcgccac ccacaaggtg gccaccggcg ccgtgagcat cgtgttcgac cagaagcagg
     3181 tgtgggtgtg caacgccgac cacgtggccg ccgccaacat cgccctgacc gtgaagggca
     3241 tcggcgagca gagcagcgac gaggagaacc ccgacggcag ccgcatcaag ctgcagctga
     3301 ccagcgctag tcatcaccat caccaccatc atcaccatca ctaggcggcc gcataatgct
     3361 taagtcgaac agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc
     3421 actataggga attcggtacc tgagaataac tagcataacc ccttggggcc tctaaacggg
     3481 tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc
     3541 cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc
     3601 ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg ccattcatcc gcttattatc
     3661 acttattcag gcgtagcaac caggcgttta agggcaccaa taactgcctt aaaaaaatta
     3721 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg
     3781 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc
     3841 ttgcgtataa tatttgccca tagtgaaaac gggggcgaag aagttgtcca tattggccac
     3901 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc
     3961 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
     4021 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc
     4081 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc
     4141 gtctttcatt gccatacgga actccggatg agcattcatc aggcgggcaa gaatgtgaat
     4201 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc
     4261 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc
     4321 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt
     4381 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
     4441 ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccaaaa
     4501 gttggcccag ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg
     4561 atcttccgtc acaggtattt attcggcgca aagtgcgtcg ggtgatgctg ccaacttact
     4621 gatttagtgt atgatggtgt ttttgaggtg ctccagtggc ttctgtttct atcagctgtc
     4681 cctcctgttc agctactgac ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc
     4741 gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct
     4801 tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata
     4861 tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa
     4921 atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa
     4981 gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg
     5041 aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt
     5101 ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca
     5161 ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca
     5221 gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta
     5281 tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca
     5341 gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac
     5401 tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt
     5461 ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca
     5521 agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata
     5581 tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg
     5641 atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg
     5701 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     5761 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5821 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     5881 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5941 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     6001 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     6061 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     6121 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     6181 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     6241 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     6301 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     6361 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     6421 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     6481 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     6541 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     6601 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     6661 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     6721 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     6781 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     6841 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     6901 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcc
//
