LOCUS       sequence-486520-        8125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of iCas12a LbCas12a with 2xSV40 nuclear 
            localization sequence for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8125)
  AUTHORS   Ma E, Chen K, Shi H, Wasko KM, Esain-Garcia I, Trinidad MI, Zhou K, 
            Ye J, Doudna JA
  TITLE     Directed evolution expands CRISPR-Cas12a genome-editing capacity.
  JOURNAL   Nucleic Acids Res. 2025 Jul 8;53(13):gkaf649. doi: 
            10.1093/nar/gkaf649.
  PUBMED    40671523
REFERENCE   2  (bases 1 to 8125)
  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: "Nucleic 
            Acids Res."; date: "2025-07-8"; volume: "53(13):gkaf649. doi"; 
            pages: " 10.1093/nar/gkaf649"
COMMENT     Sequence Label: pEM9_CL7_Bac_2NLS-iCas12a (LbCas12a)-4NLS
FEATURES             Location/Qualifiers
     source          1..8125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(75..80,81..96,97..103)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    109..125
                     /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)."
     5'UTR           147..280
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             293..310
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             722..745
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             755..775
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             785..805
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4526..4546
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4556..4576
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4586..4606
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4616..4636
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     3'UTR           4647..4791
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     terminator      4867..4938
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        5044..5148
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5149..5217,5218..6009)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5367..5386)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6180..6768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6669..6688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6940..8022)
                     /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     7984..8003
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8023..8100)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
       61 aaggaatggt gtggttgaca attaatcatc ggctcgtata atgtgtaatt gtgagcggat
      121 aacaatttga tgtgctagcg catatccagt gtagtaaggc aagtcccttc aagagttatc
      181 gttgataccc ctcgtagtgc acattccttt aacgcttcaa aatctgtaaa gcacgccata
      241 tcgccgaaag gcacacttaa ttattaagag gtaatacacc atgaaatctt ctcaccatca
      301 ccatcaccat ggttctagca aaagcaatga accgggtaaa gcaaccggtg aaggtaaacc
      361 ggttaataac aaatggctga acaatgccgg taaagatctg ggtagtccgg ttccggatcg
      421 tattgcaaat aaactgcgtg ataaagaatt cgagagcttc gatgattttc gtgaaacctt
      481 ttgggaagaa gttagcaaag atcctgaact gagcaaacag tttagccgca ataacaatga
      541 tcgtatgaaa gttggtaaag caccgaaaac acgtacccag gatgttagcg gtaaacgtac
      601 ctcatttgaa ctgaatcatc agaaaccgat tgaacagaat ggtggcgttt atgatatgga
      661 taacattagc gttgttaccc cgaaacgcaa cattgatatt gaaggcggtg gtggtggttc
      721 tttggaggtt ttgttccagg gtccaaacgc gacaccaaag aaaaagcgta aagtcggggg
      781 atctccgaaa aagaaacgga aagtaggtat ccacggagtc ccagcagcta ccatgagcaa
      841 actggagaag ttcacgaact gttattctct gtcgaaaacc ttacgtttta aggccattcc
      901 ggtaggtaaa acgcaagaaa atatcgacaa taaacgtcta ctggttgaag atgaaaagcg
      961 tgccgaagat tataaaggtg ttaaaaagct gcttgatcgc tattacctgt cttttatcaa
     1021 cgatgttctg cattccatca aattgaaaaa cttgaataat tacatctcgt tatttcgcaa
     1081 aaaaacccgt accgaaaaag aaaacaagga gctggaaaac cttgaaatta atctgcgtaa
     1141 agaaatcgcg aaagcattta aaggtaatga aggatataaa tctctgttta aaaaagacat
     1201 tattgaaacc atcctgcctg agttcttgga tgataaagat gaaattgccc tggttaatag
     1261 ctttaatggc tttacgactg catttaccgg cttttttgat aaccgtgaaa acatgttcag
     1321 cgaagaagcg aaatcgactt ccattgcctt ccgttgcatt aatgaaaacc tgacccgcta
     1381 catttctaat atggacatct ttgaaaaagt tgatgccatt ttcgacaaac atgaagttca
     1441 agaaatcaaa gaaaagattt taaactccga ttatgatgtt gaagatttct ttgagggaga
     1501 atttttcaat tttgttctca cccaagaggg tattgatgta tataacgcca ttatcggagg
     1561 atttgttacg gaatcaggcg aaaagattaa aggactgaat gaatacatta atttatacaa
     1621 tcagaaaacc aaacagaaat tgccaaaatt caaaccactg tataaacagg tattaagcga
     1681 ccgtgaatcc ttgtcctttt atggcgaagg ctatacaagc gatgaagagg tgctcgaagt
     1741 attccgtaat actttgaaca aaaactcgga aatcttctcc agcattaaaa aactggaaaa
     1801 attgtttaaa aatttcgatg aatatagtag cgcaggcatc ttcgtcaaaa atggccctgc
     1861 gatttcaacc atttctaaag acattttcgg tgaatggaac gtgatccgcg ataaatggaa
     1921 cgcggagtat gacgacattc atctgaaaaa aaaagccgtt gttaccgaaa agtatgaaga
     1981 tgaccgccgc aagagcttta aaaaaattgg tagtttctca ctggaacagt tacaagaata
     2041 tgcagacgcc gatctgtcgg ttgtggaaaa gttaaaagag attattatcc agaaagtcga
     2101 tgaaatttat aaagtgtacg gctccagcga aaagctcttt gatgccgact ttgttttaga
     2161 aaaatcgctg aaaaaaaacg atgccgtggt ggccatcatg aaagatctgt tggattcggt
     2221 caaatcgttc gaaaactata tcaaggcgtt ctttggcgaa gggaaagaaa ccaatcgtga
     2281 cgaatcgttc tatggcgatt ttgtgttggc gtatgatatt cttctgaaag tagatcacat
     2341 ctatgatgct atccgcaact acgtgaccca gaaaccgtat tcgaaggata aatttaagct
     2401 gtattttcaa aaccctcagt ttatgggtgg ctgggataaa gataaagaaa ccgactaccg
     2461 cgcgacgatc ttgcgctatg gctctaagta ctatttagcc atcatggata aaaagtatgc
     2521 aaaatgcctc cagaaaatcg ataaggatga tgtcaacggg aactacgaaa aaatcaacta
     2581 caaactcttg cctggcccga ataaaatgct gccgaaagtc tttttttcca aaaaatggat
     2641 ggcctattat aacccttccg aagatattca gaagatttac aagaacggta cctttaaaaa
     2701 aggtgacatg tttaatctga atgactgtca caagcttatt gatttcttca aagatagtat
     2761 ttctcgctac cctaaatggt caaacgcgta cgattttaat ttctcagaaa cagagaaata
     2821 caaagatatt gcggggtttt atcgcgaagt cgaagaacaa gggtataagg tgtcgttcga
     2881 atcggcttcc aaaaaggaag tggataaact tgttgaagaa ggcaaactct atatgttcca
     2941 gatctacaac aaagatttct ccgacaagag ccatggcacg ccgaacttac ataccatgta
     3001 ctttaaatta ctgttcgatg aaaataatca tggtcagatt cgcttgagcg gtggcgccga
     3061 actgtttatg cgccgcgcat cattgaaaaa ggaagaactt gttgtgcacc cagcaaacag
     3121 tccgattgca aacaaaaatc cggataaccc gaagaaaact accaccttaa gctatgatgt
     3181 atataaagat aaacgtttta gcgaagatca gtatgaactc cacattccga tcgcgattaa
     3241 caaatgtcct aagaacatct ttaaaatcaa taccgaggtt cgtgtactgc tcaaacacga
     3301 cgacaacccc tatgttattg gcattgaccg cggcgaacgc aacctgctgt atattgtcgt
     3361 tgtggatggc aaaggtaaca ttgttgaaca gtatagcctt aatgaaatca tcaataatgt
     3421 caacggcatt cgtattaaaa cggactatca tagcctgctg gacaaaaaag agaaagaacg
     3481 cttcgaagcc cgtcagaatt ggacctctat cgaaaatatc aaggaactga aagctgggta
     3541 catttctcaa gtggtccaca agatttgcga actggtggaa aagtatgacg cggtgatcgc
     3601 actggaggat ctgaattcgg gctttaaaaa tagccgtgtg aaggtggaga aacaggttta
     3661 ccaaaagttc gaaaaaatgc tgattaacaa acttaattac atggtggaca aaaaatctaa
     3721 tccatatgcg accggcgggg ctctcaaagg ctatcagatt acgaataaat tcgaaagctt
     3781 caaatccatg tctacccaga acggatttat cttttacatc cccgcctggc tgacttccaa
     3841 aattgatccg agtaccggtt ttgctaatct gctgaaaacc aaatatacgt cgatcgccga
     3901 ttcaaaaaaa ttcattagca gcttcgaccg tattatgtac gtcccggaag aagatctgtt
     3961 cgaattcgcc cttgactata aaaatttctc gcgtacggat gccgattata tcaaaaaatg
     4021 gaaactgtat agctacggca accgcatccg catttttcgc aacccaaaga agaacaacgt
     4081 atttgactgg gaggaagttt gcttgacgag cgcttacaaa gagctcttta acaaatatgg
     4141 gatcaattat cagctagggg acatccgtgt gttgctgtgt gagcagtcag ataaagcgtt
     4201 ctacagctct ttcatggctt taatgacgct gatgttgcag atgcgcaatt cgatcactgg
     4261 ccgcaccgat gtcgatttcc tgattagccc ggtcaaaaat agcgatggca ttttttacga
     4321 ttcacgtaac tatgaagccc aggaaaacgc tattctgccg aagaacgctg atgcgaacgg
     4381 cgcgtacaat attgcccgta aggtattgtg ggcaattggt cagttcaaga aagcggaaga
     4441 tgagaagttg gataaggtca aaattgcgat cccgaataaa gaatggctgg aatatgcgca
     4501 gaccagcgtg aaacatggcg gatcaccgaa gaagaaacgc aaagtcggag gtagccctaa
     4561 gaagaagcgt aaggttgggg ggtcgccaaa gaagaaacgt aaagttgggg gctcgccgaa
     4621 aaagaaacgc aaggtgtaat aggtaatctc tgcttaaaag cacagaatct aagatccctg
     4681 ccatttggcg gggatttttt tatttgtttt caggaaataa ataatcgatc gcgtaataaa
     4741 atctattatt atttttgtga agaataaatt tgggtgcaat gagaatgcgc aggccctttc
     4801 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgtcgagt aaggatctcc
     4861 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     4921 ttgtcggtga acgctctcgt cgacactatg gttgctttga cgtatgcggt gtgaaatacc
     4981 gcacagatgc gtaaggagaa aataccgcat caggcgtcag gtggcacttt tcggggaaat
     5041 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5101 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5161 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5221 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5281 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5341 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5401 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5461 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5521 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5581 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5641 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5701 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5761 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5821 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5881 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5941 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6001 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6061 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6121 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6301 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6781 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatagt catgccccgc
     6841 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     6901 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6961 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     7021 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     7081 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     7141 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     7201 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     7261 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     7321 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     7381 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     7441 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     7501 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     7561 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     7621 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     7681 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     7741 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     7801 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     7861 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     7921 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     7981 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     8041 actctcttcc gggcactatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     8101 cgggatctcg acgctctccc ttatg
//
