LOCUS       sequence-232173-        7230 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 7230)
  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 7230)
  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: BPK2014-AmCas12b
FEATURES             Location/Qualifiers
     source          1..7230
                     /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             3588..3617
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     promoter        3686..3704
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3726..3773
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3994..4653)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4568..4587
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4654..4756)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5148..5165)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5282..5826)
                     /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(5362..5381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6051..7133)
                     /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     7095..7114
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7134..7211)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cttatgcgac tcctgcatta ggaaattaat acgactcact ataggggaat tgtgagcgga
       61 taacaattcc cctgtagaaa taattttgtt taactttaat aaggagatat catatgccca
      121 agaagaagag gaaggttccc gggatgaacg tggccgtgaa gagcatcaag gtgaagctga
      181 tgctgggcca cctgcccgag atccgcgagg gcctgtggca cctgcacgag gccgtgaacc
      241 tgggcgtgcg ctactacacc gagtggctgg ccctgctgcg ccagggcaac ctgtaccgcc
      301 gcggcaagga cggcgcccag gagtgctaca tgaccgccga gcagtgccgc caggagctgc
      361 tggtgcgcct gcgcgaccgc cagaagcgca acggccacac cggcgacccc ggcaccgacg
      421 aggagctgct gggcgtggcc cgccgcctgt acgagctgct ggtgccccag agcgtgggca
      481 agaagggcca ggcccagatg ctggccagcg gcttcctgag ccccctggcc gaccccaaga
      541 gcgagggcgg caagggcacc agcaagagcg gccgcaagcc cgcctggatg ggcatgaagg
      601 aggccggcga cagccgctgg gtggaggcca aggcccgcta cgaggccaac aaggccaagg
      661 accccaccaa gcaggtgatc gccagcctgg agatgtacgg cctgcgcccc ctgttcgacg
      721 tgttcaccga gacctacaag accatccgct ggatgcccct gggcaagcac cagggcgtgc
      781 gcgcctggga ccgcgacatg ttccagcaga gcctggagcg cctgatgagc tgggagagct
      841 ggaacgagcg cgtgggcgcc gagttcgccc gcctggtgga ccgccgcgac cgcttccgcg
      901 agaagcactt caccggccag gagcacctgg tggccctggc ccagcgcctg gagcaggaga
      961 tgaaggaggc cagccccggc ttcgagagca agagcagcca ggcccaccgc atcaccaagc
     1021 gcgccctgcg cggcgccgac ggcatcatcg acgactggct gaagctgagc gagggcgagc
     1081 ccgtggaccg cttcgacgag atcctgcgca agcgccaggc ccagaacccc cgccgcttcg
     1141 gcagccacga cctgttcctg aagctggccg agcccgtgtt ccagcccctg tggcgcgagg
     1201 accccagctt cctgagccgc tgggccagct acaacgaggt gctgaacaag ctggaggacg
     1261 ccaagcagtt cgccaccttc accctgccca gcccctgcag caaccccgtg tgggcccgct
     1321 tcgagaacgc cgagggcacc aacatcttca agtacgactt cctgttcgac cacttcggca
     1381 agggccgcca cggcgtgcgc ttccagcgca tgatcgtgat gcgcgacggc gtgcccaccg
     1441 aggtggaggg catcgtggtg cccatcgccc ccagccgcca gctggacgcc ctggccccca
     1501 acgacgccgc cagccccatc gacgtgttcg tgggcgaccc cgccgccccc ggcgccttcc
     1561 gcggccagtt cggcggcgcc aagatccagt accgccgcag cgccctggtg cgcaagggcc
     1621 gccgcgagga gaaggcctac ctgtgcggct tccgcctgcc cagccagcgc cgcaccggca
     1681 cccccgccga cgacgccggc gaggtgttcc tgaacctgag cctgcgcgtg gagagccaga
     1741 gcgagcaggc cggccgccgc aaccccccct acgccgccgt gttccacatc agcgaccaga
     1801 cccgccgcgt gatcgtgcgc tacggcgaga tcgagcgcta cctggccgag caccccgaca
     1861 ccggcatccc cggcagccgc ggcctgacca gcggcctgcg cgtgatgagc gtggacctgg
     1921 gcctgcgcac cagcgccgcc atcagcgtgt tccgcgtggc ccaccgcgac gagctgaccc
     1981 ccgacgccca cggccgccag cccttcttct tccccatcca cggcatggac cacctggtgg
     2041 ccctgcacga gcgcagccac ctgatccgcc tgcccggcga gaccgagagc aagaaggtgc
     2101 gcagcatccg cgagcagcgc ctggaccgcc tgaaccgcct gcgcagccag atggccagcc
     2161 tgcgcctgct ggtgcgcacc ggcgtgctgg acgagcagaa gcgcgaccgc aactgggagc
     2221 gcctgcagag cagcatggag cgcggcggcg agcgcatgcc cagcgactgg tgggacctgt
     2281 tccaggccca ggtgcgctac ctggcccagc accgcgacgc cagcggcgag gcctggggcc
     2341 gcatggtgca ggccgccgtg cgcaccctgt ggcgccagct ggccaagcag gtgcgcgact
     2401 ggcgcaagga ggtgcgccgc aacgccgaca aggtgaagat ccgcggcatc gcccgcgacg
     2461 tgcccggcgg ccacagcctg gcccagctgg actacctgga gcgccagtac cgcttcctgc
     2521 gcagctggag cgccttcagc gtgcaggccg gccaggtggt gcgcgccgag cgcgacagcc
     2581 gcttcgccgt ggccctgcgc gagcacatcg acaacggcaa gaaggaccgc ctgaagaagc
     2641 tggccgaccg catcctgatg gaggccctgg gctacgtgta cgtgaccgac ggccgccgcg
     2701 ccggccagtg gcaggccgtg taccccccct gccagctggt gctgctggag gagctgagcg
     2761 agtaccgctt cagcaacgac cgccccccca gcgagaacag ccagctgatg gtgtggagcc
     2821 accgcggcgt gctggaggag ctgatccacc aggcccaggt gcacgacgtg ctggtgggca
     2881 ccatccccgc cgccttcagc agccgcttcg acgcccgcac cggcgccccc ggcatccgct
     2941 gccgccgcgt gcccagcatc cccctgaagg acgcccccag catccccatc tggctgagcc
     3001 actacctgaa gcagaccgag cgcgacgccg ccgccctgcg ccccggcgag ctgatcccca
     3061 ccggcgacgg cgagttcctg gtgacccccg ccggccgcgg cgccagcggc gtgcgcgtgg
     3121 tgcacgccga catcaacgcc gcccacaacc tgcagcgccg cctgtgggag aacttcgacc
     3181 tgagcgacat ccgcgtgcgc tgcgaccgcc gcgagggcaa ggacggcacc gtggtgctga
     3241 tcccccgcct gaccaaccag cgcgtgaagg agcgctacag cggcgtgatc ttcaccagcg
     3301 aggacggcgt gagcttcacc gtgggcgacg ccaagacccg ccgccgcagc agcgccagcc
     3361 agggcgaggg cgacgacctg agcgacgagg agcaggagct gctggccgag gccgacgacg
     3421 cccgcgagcg cagcgtggtg ctgttccgcg accccagcgg cttcgtgaac ggcggccgct
     3481 ggaccgccca gcgcgccttc tggggcatgg tgcacaaccg catcgagacc ctgctggccg
     3541 agcgcttcag cgtgagcggc gccgccgaga aggtgcgcgg cgctagtcat caccatcacc
     3601 accatcatca ccatcactag gcggccgcat aatgcttaag tcgaacagaa agtaatcgta
     3661 ttgtacacgg ccgcataatc gaaattaata cgactcacta tagggaattc ggtacctgag
     3721 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaac
     3781 ctcaggcatt tgagaagcac acggtcacac tgcttccggt agtcaataaa ccggtaaacc
     3841 agcaatagac ataagcggct atttaacgac cctgccctga accgacgacc gggtcgaatt
     3901 tgctttcgaa tttctgccat tcatccgctt attatcactt attcaggcgt agcaaccagg
     3961 cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc actcatcgca
     4021 gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg
     4081 aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatagt
     4141 gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact
     4201 cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc
     4261 caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc
     4321 gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta
     4381 acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaactc
     4441 cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt
     4501 atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt
     4561 acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc
     4621 aacggtggta tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct
     4681 cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc
     4741 tcttacgtgc cgatcaacgt ctcattttcg ccaaaagttg gcccagggct tcccggtatc
     4801 aacagggaca ccaggattta tttattctgc gaagtgatct tccgtcacag gtatttattc
     4861 ggcgcaaagt gcgtcgggtg atgctgccaa cttactgatt tagtgtatga tggtgttttt
     4921 gaggtgctcc agtggcttct gtttctatca gctgtccctc ctgttcagct actgacgggg
     4981 tggtgcgtaa cggcaaaagc accgccggac atcagcgcta gcggagtgta tactggctta
     5041 ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct
     5101 gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc gctcactgac
     5161 tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat
     5221 ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt
     5281 ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt
     5341 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc cctggcggct ccctcgtgcg
     5401 ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc
     5461 tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc
     5521 acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5581 acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag
     5641 ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc
     5701 gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa
     5761 ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga
     5821 tctcaagaag atcatcttat taatcagata aaatatttct agatttcagt gcaatttatc
     5881 tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag
     5941 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     6001 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     6061 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6121 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     6181 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     6241 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     6301 tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg
     6361 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     6421 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     6481 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     6541 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     6601 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     6661 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     6721 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     6781 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     6841 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     6901 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     6961 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     7021 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     7081 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     7141 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     7201 tcgatggtgt ccgggatctc gacgctctcc
//
