LOCUS       sequence-235354-        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        45..63
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    64..88
                     /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             135..224
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             231..251
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3705..3734
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     promoter        3803..3821
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3843..3890
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4111..4770)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4685..4704
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4771..4873)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5265..5282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5399..5944)
                     /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(5479..5498)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6169..7251)
                     /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     7213..7232
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7252..7329)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gggatctcga cgctctccct tatgcgactc ctgcattagg aaattaatac gactcactat
       61 aggggaattg tgagcggata acaattcccc tgtagaaata attttgttta actttaataa
      121 ggagatatca tatgtaccct tatgatgttc cagattatgc cggataccca tacgatgtcc
      181 ctgactatgc aggctcctac ccttatgacg tcccagacta cgcccccggt cccaagaaga
      241 agaggaaggt tcccgggatg gccgtgaaga gcatcaaggt gaagctgcgc ctgagcgagt
      301 gccccgacat cctggccggc atgtggcagc tgcaccgcgc caccaacgcc ggcgtgcgct
      361 actacaccga gtgggtgagc ctgatgcgcc aggagatcct gtacagccgc ggccccgacg
      421 gcggccagca gtgctacatg accgccgagg actgccagcg cgagctgctg cgccgcctgc
      481 gcaaccgcca gctgcacaac ggccgccagg accagcccgg caccgacgcc gacctgctgg
      541 ccatcagccg ccgcctgtac gagatcctgg tgctgcagag catcggcaag cgcggcgacg
      601 cccagcagat cgccagcagc ttcctgagcc ccctggtgga ccccaacagc aagggcggcc
      661 gcggcgaggc caagagcggc cgcaagcccg cctggcagaa gatgcgcgac cagggcgacc
      721 cccgctgggt ggccgcccgc gagaagtacg agcagcgcaa ggccgtggac cccagcaagg
      781 agatcctgaa cagcctggac gccctgggcc tgcgccccct gttcgccgtg ttcaccgaga
      841 cctaccgcag cggcgtggac tggaagcccc tgggcaagag ccagggcgtg cgcacctggg
      901 accgcgacat gttccagcag gccctggagc gcctgatgag ctgggagagc tggaaccgcc
      961 gcgtgggcga ggagtacgcc cgcctgttcc agcagaagat gaagttcgag caggagcact
     1021 tcgccgagca gagccacctg gtgaagctgg cccgcgccct ggaggccgac atgcgcgccg
     1081 ccagccaggg cttcgaggcc aagcgcggca ccgcccacca gatcacccgc cgcgccctgc
     1141 gcggcgccga ccgcgtgttc gagatctgga agagcatccc cgaggaggcc ctgttcagcc
     1201 agtacgacga ggtgatccgc caggtgcagg ccgagaagcg ccgcgacttc ggcagccacg
     1261 acctgttcgc caagctggcc gagcccaagt accagcccct gtggcgcgcc gacgagacct
     1321 tcctgacccg ctacgccctg tacaacggcg tgctgcgcga cctggagaag gcccgccagt
     1381 tcgccacctt caccctgccc gacgcctgcg tgaaccccat ctggacccgc ttcgagagca
     1441 gccagggcag caacctgcac aagtacgagt tcctgttcga ccacctgggc cccggccgcc
     1501 acgccgtgcg cttccagcgc ctgctggtgg tggagagcga gggcgccaag gagcgcgaca
     1561 gcgtggtggt gcccgtggcc cccagcggcc agctggacaa gctggtgctg cgcgaggagg
     1621 agaagagcag cgtggccctg cacctgcacg acaccgcccg ccccgacggc ttcatggccg
     1681 agtgggccgg cgccaagctg cagtacgagc gcagcaccct ggcccgcaag gcccgccgcg
     1741 acaagcaggg catgcgcagc tggcgccgcc agcccagcat gctgatgagc gccgcccaga
     1801 tgctggagga cgccaagcag gccggcgacg tgtacctgaa catcagcgtg cgcgtgaaga
     1861 gccccagcga ggtgcgcggc cagcgccgcc ccccctacgc cgccctgttc cgcatcgacg
     1921 acaagcagcg ccgcgtgacc gtgaactaca acaagctgag cgcctacctg gaggagcacc
     1981 ccgacaagca gatccccggc gcccccggcc tgctgagcgg cctgcgcgtg atgagcgtgg
     2041 acctgggcct gcgcaccagc gccagcatca gcgtgttccg cgtggccaag aaggaggagg
     2101 tggaggccct gggcgacggc cgcccccccc actactaccc catccacggc accgacgacc
     2161 tggtggccgt gcacgagcgc agccacctga tccagatgcc cggcgagacc gagaccaagc
     2221 agctgcgcaa gctgcgcgag gagcgccagg ccgtgctgcg ccccctgttc gcccagctgg
     2281 ccctgctgcg cctgctggtg cgctgcggcg ccgccgacga gcgcatccgc acccgcagct
     2341 ggcagcgcct gaccaagcag ggccgcgagt tcaccaagcg cctgaccccc agctggcgcg
     2401 aggccctgga gctggagctg acccgcctgg aggcctactg cggccgcgtg cccgacgacg
     2461 agtggagccg catcgtggac cgcaccgtga tcgccctgtg gcgccgcatg ggcaagcagg
     2521 tgcgcgactg gcgcaagcag gtgaagagcg gcgccaaggt gaaggtgaag ggctaccagc
     2581 tggacgtggt gggcggcaac agcctggccc agatcgacta cctggagcag cagtacaagt
     2641 tcctgcgccg ctggagcttc ttcgcccgcg ccagcggcct ggtggtgcgc gccgaccgcg
     2701 agagccactt cgccgtggcc ctgcgccagc acatcgagaa cgccaagcgc gaccgcctga
     2761 agaagctggc cgaccgcatc ctgatggagg ccctgggcta cgtgtacgag gccagcggcc
     2821 cccgcgaggg ccagtggacc gcccagcacc ccccctgcca gctgatcatc ctggaggagc
     2881 tgagcgccta ccgcttcagc gacgaccgcc cccccagcga gaacagcaag ctgatggcct
     2941 ggggccaccg cggcatcctg gaggagctgg tgaaccaggc ccaggtgcac gacgtgctgg
     3001 tgggcaccgt gtacgccgcc ttcagcagcc gcttcgacgc ccgcaccggc gcccccggcg
     3061 tgcgctgccg ccgcgtgccc gcccgcttcg tgggcgccac cgtggacgac agcctgcccc
     3121 tgtggctgac cgagttcctg gacaagcacc gcctggacaa gaacctgctg cgccccgacg
     3181 acgtgatccc caccggcgag ggcgagttcc tggtgagccc ctgcggcgag gaggccgccc
     3241 gcgtgcgcca ggtgcacgcc gacatcaacg ccgcccagaa cctgcagcgc cgcctgtggc
     3301 agaacttcga catcaccgag ctgcgcctgc gctgcgacgt gaagatgggc ggcgagggca
     3361 ccgtgctggt gccccgcgtg aacaacgccc gcgccaagca gctgttcggc aagaaggtgc
     3421 tggtgagcca ggacggcgtg accttcttcg agcgcagcca gaccggcggc aagccccaca
     3481 gcgagaagca gaccgacctg accgacaagg agctggagct gatcgccgag gccgacgagg
     3541 cccgcgccaa gagcgtggtg ctgttccgcg accccagcgg ccacatcggc aagggccact
     3601 ggatccgcca gcgcgagttc tggagcctgg tgaagcagcg catcgagagc cacaccgccg
     3661 agcgcatccg cgtgcgcggc gtgggcagca gcctggacgc tagtcatcac catcaccacc
     3721 atcatcacca tcactaggcg gccgcataat gcttaagtcg aacagaaagt aatcgtattg
     3781 tacacggccg cataatcgaa attaatacga ctcactatag ggaattcggt acctgagaat
     3841 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaacctc
     3901 aggcatttga gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc
     3961 aatagacata agcggctatt taacgaccct gccctgaacc gacgaccggg tcgaatttgc
     4021 tttcgaattt ctgccattca tccgcttatt atcacttatt caggcgtagc aaccaggcgt
     4081 ttaagggcac caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta
     4141 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
     4201 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatagtgaa
     4261 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     4321 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     4381 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     4441 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     4501 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac ggaactccgg
     4561 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     4621 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     4681 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     4741 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     4801 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     4861 tacgtgccga tcaacgtctc attttcgcca aaagttggcc cagggcttcc cggtatcaac
     4921 agggacacca ggatttattt attctgcgaa gtgatcttcc gtcacaggta tttattcggc
     4981 gcaaagtgcg tcgggtgatg ctgccaactt actgatttag tgtatgatgg tgtttttgag
     5041 gtgctccagt ggcttctgtt tctatcagct gtccctcctg ttcagctact gacggggtgg
     5101 tgcgtaacgg caaaagcacc gccggacatc agcgctagcg gagtgtatac tggcttacta
     5161 tgttggcact gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca
     5221 ccggtgcgtc agcagaatat gtgatacagg atatattccg cttcctcgct cactgactcg
     5281 ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc
     5341 ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt
     5401 tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc
     5461 gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct
     5521 ctcctgttcc tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc
     5581 attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac
     5641 gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5701 ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt
     5761 agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc
     5821 tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc
     5881 gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc
     5941 tcaagaagat catcttatta atcagataaa atatttctag atttcagtgc aatttatctc
     6001 ttcaaatgta gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc
     6061 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     6121 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     6181 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6241 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     6301 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     6361 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     6421 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     6481 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     6541 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6601 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6661 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6721 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6781 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6841 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6901 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6961 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     7021 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     7081 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     7141 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     7201 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     7261 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     7321 gatggtgtcc
//
