LOCUS       sequence-235339-        7213 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression, protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7213)
  AUTHORS   Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q, Li T, Li J, Zhou Q, Li W
  TITLE     Repurposing CRISPR-Cas12b for mammalian genome engineering.
  JOURNAL   Cell Discov. 2018 Nov 27;4:63. doi: 10.1038/s41421-018-0069-3. 
            eCollection 2018.
  PUBMED    30510770
REFERENCE   2  (bases 1 to 7213)
  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: "Cell 
            Discov."; date: "2018-11-27"; volume: "4:63. doi"; pages: " 
            10.1038/s41421-018-0069-3. eCollection 2018"
COMMENT     Sequence Label: BPK2013-BsCas12b
FEATURES             Location/Qualifiers
     source          1..7213
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..22
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    23..47
                     /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             94..183
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             190..210
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3547..3576
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     promoter        3645..3663
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3685..3732
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3953..4612)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4527..4546
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4613..4715)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5107..5124)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5241..5786)
                     /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(5321..5340)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6011..7093)
                     /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(7094..7171)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aattaatacg actcactata ggggaattgt gagcggataa caattcccct gtagaaataa
       61 ttttgtttaa ctttaataag gagatatcat atgtaccctt atgatgttcc agattatgcc
      121 ggatacccat acgatgtccc tgactatgca ggctcctacc cttatgacgt cccagactac
      181 gcccccggtc ccaagaagaa gaggaaggtt cccgggatgg ccatccgcag catcaagctg
      241 aagctgaaga cccacaccgg ccccgaggcc cagaacctgc gcaagggcat ctggcgcacc
      301 caccgcctgc tgaacgaggg cgtggcctac tacatgaaga tgctgctgct gttccgccag
      361 gagagcaccg gcgagcgccc caaggaggag ctgcaggagg agctgatctg ccacatccgc
      421 gagcagcagc agcgcaacca ggccgacaag aacacccagg ccctgcccct ggacaaggcc
      481 ctggaggccc tgcgccagct gtacgagctg ctggtgccca gcagcgtggg ccagagcggc
      541 gacgcccaga tcatcagccg caagttcctg agccccctgg tggaccccaa cagcgagggc
      601 ggcaagggca ccagcaaggc cggcgccaag cccacctggc agaagaagaa ggaggccaac
      661 gaccccacct gggagcagga ctacgagaag tggaagaagc gccgcgagga ggaccccacc
      721 gccagcgtga tcaccaccct ggaggagtac ggcatccgcc ccatcttccc cctgtacacc
      781 aacaccgtga ccgacatcgc ctggctgccc ctgcagagca accagttcgt gcgcacctgg
      841 gaccgcgaca tgctgcagca ggccatcgag cgcctgctga gctgggagag ctggaacaag
      901 cgcgtgcagg aggagtacgc caagctgaag gagaagatgg cccagctgaa cgagcagctg
      961 gagggcggcc aggagtggat cagcctgctg gagcagtacg aggagaaccg cgagcgcgag
     1021 ctgcgcgaga acatgaccgc cgccaacgac aagtaccgca tcaccaagcg ccagatgaag
     1081 ggctggaacg agctgtacga gctgtggagc accttccccg ccagcgccag ccacgagcag
     1141 tacaaggagg ccctgaagcg cgtgcagcag cgcctgcgcg gccgcttcgg cgacgcccac
     1201 ttcttccagt acctgatgga ggagaagaac cgcctgatct ggaagggcaa cccccagcgc
     1261 atccactact tcgtggcccg caacgagctg accaagcgcc tggaggaggc caagcagagc
     1321 gccaccatga ccctgcccaa cgcccgcaag caccccctgt gggtgcgctt cgacgcccgc
     1381 ggcggcaacc tgcaggacta ctacctgacc gccgaggccg acaagccccg cagccgccgc
     1441 ttcgtgacct tcagccagct gatctggccc agcgagagcg gctggatgga gaagaaggac
     1501 gtggaggtgg agctggccct gagccgccag ttctaccagc aggtgaagct gctgaagaac
     1561 gacaagggca agcagaagat cgagttcaag gacaagggca gcggcagcac cttcaacggc
     1621 cacctgggcg gcgccaagct gcagctggag cgcggcgacc tggagaagga ggagaagaac
     1681 ttcgaggacg gcgagatcgg cagcgtgtac ctgaacgtgg tgatcgactt cgagcccctg
     1741 caggaggtga agaacggccg cgtgcaggcc ccctacggcc aggtgctgca gctgatccgc
     1801 cgccccaacg agttccccaa ggtgaccacc tacaagagcg agcagctggt ggagtggatc
     1861 aaggccagcc cccagcacag cgccggcgtg gagagcctgg ccagcggctt ccgcgtgatg
     1921 agcatcgacc tgggcctgcg cgccgccgcc gccaccagca tcttcagcgt ggaggagagc
     1981 agcgacaaga acgccgccga cttcagctac tggatcgagg gcacccccct ggtggccgtg
     2041 caccagcgca gctacatgct gcgcctgccc ggcgagcagg tggagaagca ggtgatggag
     2101 aagcgcgacg agcgcttcca gctgcaccag cgcgtgaagt tccagatccg cgtgctggcc
     2161 cagatcatgc gcatggccaa caagcagtac ggcgaccgct gggacgagct ggacagcctg
     2221 aagcaggccg tggagcagaa gaagagcccc ctggaccaga ccgaccgcac cttctgggag
     2281 ggcatcgtgt gcgacctgac caaggtgctg ccccgcaacg aggccgactg ggagcaggcc
     2341 gtggtgcaga tccaccgcaa ggccgaggag tacgtgggca aggccgtgca ggcctggcgc
     2401 aagcgcttcg ccgccgacga gcgcaagggc atcgccggcc tgagcatgtg gaacatcgag
     2461 gagctggagg gcctgcgcaa gctgctgatc agctggagcc gccgcacccg caacccccag
     2521 gaggtgaacc gcttcgagcg cggccacacc agccaccagc gcctgctgac ccacatccag
     2581 aacgtgaagg aggaccgcct gaagcagctg agccacgcca tcgtgatgac cgccctgggc
     2641 tacgtgtacg acgagcgcaa gcaggagtgg tgcgccgagt accccgcctg ccaggtgatc
     2701 ctgttcgaga acctgagcca gtaccgcagc aacctggacc gcagcaccaa ggagaacagc
     2761 accctgatga agtgggccca ccgcagcatc cccaagtacg tgcacatgca ggccgagccc
     2821 tacggcatcc agatcggcga cgtgcgcgcc gagtacagca gccgcttcta cgccaagacc
     2881 ggcacccccg gcatccgctg caagaaggtg cgcggccagg acctgcaggg ccgccgcttc
     2941 gagaacctgc agaagcgcct ggtgaacgag cagttcctga ccgaggagca ggtgaagcag
     3001 ctgcgccccg gcgacatcgt gcccgacgac agcggcgagc tgttcatgac cctgaccgac
     3061 ggcagcggca gcaaggaggt ggtgttcctg caggccgaca tcaacgccgc ccacaacctg
     3121 cagaagcgct tctggcagcg ctacaacgag ctgttcaagg tgagctgccg cgtgatcgtg
     3181 cgcgacgagg aggagtacct ggtgcccaag accaagagcg tgcaggccaa gctgggcaag
     3241 ggcctgttcg tgaagaagag cgacaccgcc tggaaggacg tgtacgtgtg ggacagccag
     3301 gccaagctga agggcaagac caccttcacc gaggagagcg agagccccga gcagctggag
     3361 gacttccagg agatcatcga ggaggccgag gaggccaagg gcacctaccg caccctgttc
     3421 cgcgacccca gcggcgtgtt cttccccgag agcgtgtggt acccccagaa ggacttctgg
     3481 ggcgaggtga agcgcaagct gtacggcaag ctgcgcgagc gcttcctgac caaggcccgc
     3541 gctagtcatc accatcacca ccatcatcac catcactagg cggccgcata atgcttaagt
     3601 cgaacagaaa gtaatcgtat tgtacacggc cgcataatcg aaattaatac gactcactat
     3661 agggaattcg gtacctgaga ataactagca taaccccttg gggcctctaa acgggtcttg
     3721 aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact gcttccggta
     3781 gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc ctgccctgaa
     3841 ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt catccgctta ttatcactta
     3901 ttcaggcgta gcaaccaggc gtttaagggc accaataact gccttaaaaa aattacgccc
     3961 cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc
     4021 catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg
     4081 tataatattt gcccatagtg aaaacggggg cgaagaagtt gtccatattg gccacgttta
     4141 aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa
     4201 accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt
     4261 gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt
     4321 gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt
     4381 tcattgccat acggaactcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg
     4441 ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct
     4501 gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac
     4561 gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt
     4621 ccttagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat
     4681 tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg
     4741 cccagggctt cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt
     4801 ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac ttactgattt
     4861 agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag ctgtccctcc
     4921 tgttcagcta ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctag
     4981 cggagtgtat actggcttac tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg
     5041 tggcaggaga aaaaaggctg caccggtgcg tcagcagaat atgtgataca ggatatattc
     5101 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc
     5161 ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag
     5221 agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc
     5281 tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5341 cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg
     5401 ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg
     5461 ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg
     5521 taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac
     5581 tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa
     5641 ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag
     5701 ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga
     5761 ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct
     5821 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     5881 agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     5941 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     6001 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6061 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     6121 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     6181 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     6241 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     6301 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     6361 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     6421 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     6481 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     6541 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     6601 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     6661 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     6721 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     6781 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     6841 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     6901 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     6961 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     7021 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     7081 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     7141 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     7201 tcctgcatta gga
//
