LOCUS       sequence-232172-        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        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             3570..3599
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     promoter        3668..3686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3709..3756
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3977..4636)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4551..4570
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4637..4739)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5131..5148)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5265..5809)
                     /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(5345..5364)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6034..7116)
                     /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(7117..7194)
                     /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 tggccatccg cagcatcaag ctgaagctga agacccacac cggccccgag gcccagaacc
      301 tgcgcaaggg catctggcgc acccaccgcc tgctgaacga gggcgtggcc tactacatga
      361 agatgctgct gctgttccgc caggagagca ccggcgagcg ccccaaggag gagctgcagg
      421 aggagctgat ctgccacatc cgcgagcagc agcagcgcaa ccaggccgac aagaacaccc
      481 aggccctgcc cctggacaag gccctggagg ccctgcgcca gctgtacgag ctgctggtgc
      541 ccagcagcgt gggccagagc ggcgacgccc agatcatcag ccgcaagttc ctgagccccc
      601 tggtggaccc caacagcgag ggcggcaagg gcaccagcaa ggccggcgcc aagcccacct
      661 ggcagaagaa gaaggaggcc aacgacccca cctgggagca ggactacgag aagtggaaga
      721 agcgccgcga ggaggacccc accgccagcg tgatcaccac cctggaggag tacggcatcc
      781 gccccatctt ccccctgtac accaacaccg tgaccgacat cgcctggctg cccctgcaga
      841 gcaaccagtt cgtgcgcacc tgggaccgcg acatgctgca gcaggccatc gagcgcctgc
      901 tgagctggga gagctggaac aagcgcgtgc aggaggagta cgccaagctg aaggagaaga
      961 tggcccagct gaacgagcag ctggagggcg gccaggagtg gatcagcctg ctggagcagt
     1021 acgaggagaa ccgcgagcgc gagctgcgcg agaacatgac cgccgccaac gacaagtacc
     1081 gcatcaccaa gcgccagatg aagggctgga acgagctgta cgagctgtgg agcaccttcc
     1141 ccgccagcgc cagccacgag cagtacaagg aggccctgaa gcgcgtgcag cagcgcctgc
     1201 gcggccgctt cggcgacgcc cacttcttcc agtacctgat ggaggagaag aaccgcctga
     1261 tctggaaggg caacccccag cgcatccact acttcgtggc ccgcaacgag ctgaccaagc
     1321 gcctggagga ggccaagcag agcgccacca tgaccctgcc caacgcccgc aagcaccccc
     1381 tgtgggtgcg cttcgacgcc cgcggcggca acctgcagga ctactacctg accgccgagg
     1441 ccgacaagcc ccgcagccgc cgcttcgtga ccttcagcca gctgatctgg cccagcgaga
     1501 gcggctggat ggagaagaag gacgtggagg tggagctggc cctgagccgc cagttctacc
     1561 agcaggtgaa gctgctgaag aacgacaagg gcaagcagaa gatcgagttc aaggacaagg
     1621 gcagcggcag caccttcaac ggccacctgg gcggcgccaa gctgcagctg gagcgcggcg
     1681 acctggagaa ggaggagaag aacttcgagg acggcgagat cggcagcgtg tacctgaacg
     1741 tggtgatcga cttcgagccc ctgcaggagg tgaagaacgg ccgcgtgcag gccccctacg
     1801 gccaggtgct gcagctgatc cgccgcccca acgagttccc caaggtgacc acctacaaga
     1861 gcgagcagct ggtggagtgg atcaaggcca gcccccagca cagcgccggc gtggagagcc
     1921 tggccagcgg cttccgcgtg atgagcatcg acctgggcct gcgcgccgcc gccgccacca
     1981 gcatcttcag cgtggaggag agcagcgaca agaacgccgc cgacttcagc tactggatcg
     2041 agggcacccc cctggtggcc gtgcaccagc gcagctacat gctgcgcctg cccggcgagc
     2101 aggtggagaa gcaggtgatg gagaagcgcg acgagcgctt ccagctgcac cagcgcgtga
     2161 agttccagat ccgcgtgctg gcccagatca tgcgcatggc caacaagcag tacggcgacc
     2221 gctgggacga gctggacagc ctgaagcagg ccgtggagca gaagaagagc cccctggacc
     2281 agaccgaccg caccttctgg gagggcatcg tgtgcgacct gaccaaggtg ctgccccgca
     2341 acgaggccga ctgggagcag gccgtggtgc agatccaccg caaggccgag gagtacgtgg
     2401 gcaaggccgt gcaggcctgg cgcaagcgct tcgccgccga cgagcgcaag ggcatcgccg
     2461 gcctgagcat gtggaacatc gaggagctgg agggcctgcg caagctgctg atcagctgga
     2521 gccgccgcac ccgcaacccc caggaggtga accgcttcga gcgcggccac accagccacc
     2581 agcgcctgct gacccacatc cagaacgtga aggaggaccg cctgaagcag ctgagccacg
     2641 ccatcgtgat gaccgccctg ggctacgtgt acgacgagcg caagcaggag tggtgcgccg
     2701 agtaccccgc ctgccaggtg atcctgttcg agaacctgag ccagtaccgc agcaacctgg
     2761 accgcagcac caaggagaac agcaccctga tgaagtgggc ccaccgcagc atccccaagt
     2821 acgtgcacat gcaggccgag ccctacggca tccagatcgg cgacgtgcgc gccgagtaca
     2881 gcagccgctt ctacgccaag accggcaccc ccggcatccg ctgcaagaag gtgcgcggcc
     2941 aggacctgca gggccgccgc ttcgagaacc tgcagaagcg cctggtgaac gagcagttcc
     3001 tgaccgagga gcaggtgaag cagctgcgcc ccggcgacat cgtgcccgac gacagcggcg
     3061 agctgttcat gaccctgacc gacggcagcg gcagcaagga ggtggtgttc ctgcaggccg
     3121 acatcaacgc cgcccacaac ctgcagaagc gcttctggca gcgctacaac gagctgttca
     3181 aggtgagctg ccgcgtgatc gtgcgcgacg aggaggagta cctggtgccc aagaccaaga
     3241 gcgtgcaggc caagctgggc aagggcctgt tcgtgaagaa gagcgacacc gcctggaagg
     3301 acgtgtacgt gtgggacagc caggccaagc tgaagggcaa gaccaccttc accgaggaga
     3361 gcgagagccc cgagcagctg gaggacttcc aggagatcat cgaggaggcc gaggaggcca
     3421 agggcaccta ccgcaccctg ttccgcgacc ccagcggcgt gttcttcccc gagagcgtgt
     3481 ggtaccccca gaaggacttc tggggcgagg tgaagcgcaa gctgtacggc aagctgcgcg
     3541 agcgcttcct gaccaaggcc cgcgctagtc atcaccatca ccaccatcat caccatcact
     3601 aggcggccgc ataatgctta agtcgaacag aaagtaatcg tattgtacac ggccgcataa
     3661 tcgaaattaa tacgactcac tatagggaat tcggtacctg agcaataact agcataaccc
     3721 cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag
     3781 cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg
     3841 gctatttaac gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc
     3901 cattcatccg cttattatca cttattcagg cgtagcaacc aggcgtttaa gggcaccaat
     3961 aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat
     4021 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
     4081 gcatcagcac cttgtcgcct tgcgtataat atttgcccat agtgaaaacg ggggcgaaga
     4141 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
     4201 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
     4261 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc
     4321 agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
     4381 cccatatcac cagctcaccg tctttcattg ccatacggaa ctccggatga gcattcatca
     4441 ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct
     4501 ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact
     4561 gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag
     4621 tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
     4681 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     4741 cgtctcattt tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat
     4801 ttatttattc tgcgaagtga tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg
     4861 gtgatgctgc caacttactg atttagtgta tgatggtgtt tttgaggtgc tccagtggct
     4921 tctgtttcta tcagctgtcc ctcctgttca gctactgacg gggtggtgcg taacggcaaa
     4981 agcaccgccg gacatcagcg ctagcggagt gtatactggc ttactatgtt ggcactgatg
     5041 agggtgtcag tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca
     5101 gaatatgtga tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt
     5161 tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag
     5221 gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc
     5281 ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga
     5341 ctataaagat accaggcgtt tcccctggcg gctccctcgt gcgctctcct gttcctgcct
     5401 ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca
     5461 ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag
     5521 tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca
     5581 aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg
     5641 cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt
     5701 acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt
     5761 tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct
     5821 tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc
     5881 tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac
     5941 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     6001 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6061 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6121 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     6181 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     6241 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     6301 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     6361 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     6421 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     6481 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     6541 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     6601 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     6661 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     6721 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     6781 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     6841 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     6901 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     6961 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     7021 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     7081 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     7141 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     7201 ctcgacgctc tcc
//
