LOCUS       sequence-232174-        7330 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 7330)
  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 7330)
  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-AkCas12b
FEATURES             Location/Qualifiers
     source          1..7330
                     /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..206
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             213..233
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3687..3716
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     promoter        3785..3803
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3826..3873
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4094..4753)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4668..4687
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4754..4856)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5248..5265)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5382..5926)
                     /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(5462..5481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6151..7233)
                     /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"
     primer_bind     7195..7214
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7234..7311)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cttatgcgac tcctgcatta ggaaattaat acgactcact ataggggaat tgtgagcgga
       61 taacaattcc cctgtagaaa taattttgtt taactttaat aaggagatat catatgtacc
      121 cttatgatgt tccagattat gccggatacc catacgatgt ccctgactat gcaggctcct
      181 acccttatga cgtcccagac tacgcccccg gtcccaagaa gaagaggaag gttcccggga
      241 tggccgtgaa gagcatcaag gtgaagctgc gcctgagcga gtgccccgac atcctggccg
      301 gcatgtggca gctgcaccgc gccaccaacg ccggcgtgcg ctactacacc gagtgggtga
      361 gcctgatgcg ccaggagatc ctgtacagcc gcggccccga cggcggccag cagtgctaca
      421 tgaccgccga ggactgccag cgcgagctgc tgcgccgcct gcgcaaccgc cagctgcaca
      481 acggccgcca ggaccagccc ggcaccgacg ccgacctgct ggccatcagc cgccgcctgt
      541 acgagatcct ggtgctgcag agcatcggca agcgcggcga cgcccagcag atcgccagca
      601 gcttcctgag ccccctggtg gaccccaaca gcaagggcgg ccgcggcgag gccaagagcg
      661 gccgcaagcc cgcctggcag aagatgcgcg accagggcga cccccgctgg gtggccgccc
      721 gcgagaagta cgagcagcgc aaggccgtgg accccagcaa ggagatcctg aacagcctgg
      781 acgccctggg cctgcgcccc ctgttcgccg tgttcaccga gacctaccgc agcggcgtgg
      841 actggaagcc cctgggcaag agccagggcg tgcgcacctg ggaccgcgac atgttccagc
      901 aggccctgga gcgcctgatg agctgggaga gctggaaccg ccgcgtgggc gaggagtacg
      961 cccgcctgtt ccagcagaag atgaagttcg agcaggagca cttcgccgag cagagccacc
     1021 tggtgaagct ggcccgcgcc ctggaggccg acatgcgcgc cgccagccag ggcttcgagg
     1081 ccaagcgcgg caccgcccac cagatcaccc gccgcgccct gcgcggcgcc gaccgcgtgt
     1141 tcgagatctg gaagagcatc cccgaggagg ccctgttcag ccagtacgac gaggtgatcc
     1201 gccaggtgca ggccgagaag cgccgcgact tcggcagcca cgacctgttc gccaagctgg
     1261 ccgagcccaa gtaccagccc ctgtggcgcg ccgacgagac cttcctgacc cgctacgccc
     1321 tgtacaacgg cgtgctgcgc gacctggaga aggcccgcca gttcgccacc ttcaccctgc
     1381 ccgacgcctg cgtgaacccc atctggaccc gcttcgagag cagccagggc agcaacctgc
     1441 acaagtacga gttcctgttc gaccacctgg gccccggccg ccacgccgtg cgcttccagc
     1501 gcctgctggt ggtggagagc gagggcgcca aggagcgcga cagcgtggtg gtgcccgtgg
     1561 cccccagcgg ccagctggac aagctggtgc tgcgcgagga ggagaagagc agcgtggccc
     1621 tgcacctgca cgacaccgcc cgccccgacg gcttcatggc cgagtgggcc ggcgccaagc
     1681 tgcagtacga gcgcagcacc ctggcccgca aggcccgccg cgacaagcag ggcatgcgca
     1741 gctggcgccg ccagcccagc atgctgatga gcgccgccca gatgctggag gacgccaagc
     1801 aggccggcga cgtgtacctg aacatcagcg tgcgcgtgaa gagccccagc gaggtgcgcg
     1861 gccagcgccg ccccccctac gccgccctgt tccgcatcga cgacaagcag cgccgcgtga
     1921 ccgtgaacta caacaagctg agcgcctacc tggaggagca ccccgacaag cagatccccg
     1981 gcgcccccgg cctgctgagc ggcctgcgcg tgatgagcgt ggacctgggc ctgcgcacca
     2041 gcgccagcat cagcgtgttc cgcgtggcca agaaggagga ggtggaggcc ctgggcgacg
     2101 gccgcccccc ccactactac cccatccacg gcaccgacga cctggtggcc gtgcacgagc
     2161 gcagccacct gatccagatg cccggcgaga ccgagaccaa gcagctgcgc aagctgcgcg
     2221 aggagcgcca ggccgtgctg cgccccctgt tcgcccagct ggccctgctg cgcctgctgg
     2281 tgcgctgcgg cgccgccgac gagcgcatcc gcacccgcag ctggcagcgc ctgaccaagc
     2341 agggccgcga gttcaccaag cgcctgaccc ccagctggcg cgaggccctg gagctggagc
     2401 tgacccgcct ggaggcctac tgcggccgcg tgcccgacga cgagtggagc cgcatcgtgg
     2461 accgcaccgt gatcgccctg tggcgccgca tgggcaagca ggtgcgcgac tggcgcaagc
     2521 aggtgaagag cggcgccaag gtgaaggtga agggctacca gctggacgtg gtgggcggca
     2581 acagcctggc ccagatcgac tacctggagc agcagtacaa gttcctgcgc cgctggagct
     2641 tcttcgcccg cgccagcggc ctggtggtgc gcgccgaccg cgagagccac ttcgccgtgg
     2701 ccctgcgcca gcacatcgag aacgccaagc gcgaccgcct gaagaagctg gccgaccgca
     2761 tcctgatgga ggccctgggc tacgtgtacg aggccagcgg cccccgcgag ggccagtgga
     2821 ccgcccagca ccccccctgc cagctgatca tcctggagga gctgagcgcc taccgcttca
     2881 gcgacgaccg cccccccagc gagaacagca agctgatggc ctggggccac cgcggcatcc
     2941 tggaggagct ggtgaaccag gcccaggtgc acgacgtgct ggtgggcacc gtgtacgccg
     3001 ccttcagcag ccgcttcgac gcccgcaccg gcgcccccgg cgtgcgctgc cgccgcgtgc
     3061 ccgcccgctt cgtgggcgcc accgtggacg acagcctgcc cctgtggctg accgagttcc
     3121 tggacaagca ccgcctggac aagaacctgc tgcgccccga cgacgtgatc cccaccggcg
     3181 agggcgagtt cctggtgagc ccctgcggcg aggaggccgc ccgcgtgcgc caggtgcacg
     3241 ccgacatcaa cgccgcccag aacctgcagc gccgcctgtg gcagaacttc gacatcaccg
     3301 agctgcgcct gcgctgcgac gtgaagatgg gcggcgaggg caccgtgctg gtgccccgcg
     3361 tgaacaacgc ccgcgccaag cagctgttcg gcaagaaggt gctggtgagc caggacggcg
     3421 tgaccttctt cgagcgcagc cagaccggcg gcaagcccca cagcgagaag cagaccgacc
     3481 tgaccgacaa ggagctggag ctgatcgccg aggccgacga ggcccgcgcc aagagcgtgg
     3541 tgctgttccg cgaccccagc ggccacatcg gcaagggcca ctggatccgc cagcgcgagt
     3601 tctggagcct ggtgaagcag cgcatcgaga gccacaccgc cgagcgcatc cgcgtgcgcg
     3661 gcgtgggcag cagcctggac gctagtcatc accatcacca ccatcatcac catcactagg
     3721 cggccgcata atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc cgcataatcg
     3781 aaattaatac gactcactat agggaattcg gtacctgagc aataactagc ataacccctt
     3841 ggggcctcta aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac
     3901 acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct
     3961 atttaacgac cctgccctga accgacgacc gggtcgaatt tgctttcgaa tttctgccat
     4021 tcatccgctt attatcactt attcaggcgt agcaaccagg cgtttaaggg caccaataac
     4081 tgccttaaaa aaattacgcc ccgccctgcc actcatcgca gtactgttgt aattcattaa
     4141 gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca
     4201 tcagcacctt gtcgccttgc gtataatatt tgcccatagt gaaaacgggg gcgaagaagt
     4261 tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga
     4321 cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg
     4381 ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga
     4441 gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc
     4501 atatcaccag ctcaccgtct ttcattgcca tacggaactc cggatgagca ttcatcaggc
     4561 gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta
     4621 aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa
     4681 atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga
     4741 tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc
     4801 ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt
     4861 ctcattttcg ccaaaagttg gcccagggct tcccggtatc aacagggaca ccaggattta
     4921 tttattctgc gaagtgatct tccgtcacag gtatttattc ggcgcaaagt gcgtcgggtg
     4981 atgctgccaa cttactgatt tagtgtatga tggtgttttt gaggtgctcc agtggcttct
     5041 gtttctatca gctgtccctc ctgttcagct actgacgggg tggtgcgtaa cggcaaaagc
     5101 accgccggac atcagcgcta gcggagtgta tactggctta ctatgttggc actgatgagg
     5161 gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc gtcagcagaa
     5221 tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg
     5281 actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa
     5341 gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc
     5401 cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta
     5461 taaagatacc aggcgtttcc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc
     5521 ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc
     5581 agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc
     5641 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa
     5701 gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc
     5761 cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc
     5821 tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt
     5881 ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat
     5941 taatcagata aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     6001 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     6061 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     6121 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6181 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6241 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     6301 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     6361 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     6421 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     6481 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     6541 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     6601 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     6661 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     6721 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     6781 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     6841 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     6901 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     6961 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     7021 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     7081 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     7141 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     7201 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     7261 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     7321 gacgctctcc
//
