LOCUS       sequence-486525-        8125 bp    DNA     circular SYN 17-SEP-2025
DEFINITION  Bacterial expression of Flex-Cas12a LbCas12a with 2xSV40 NLS at 
            N-terminus and 4xSV40 at C-terminus.
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 Sep 17, 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: pEM7_CL7_Bac_2NLS-Flex-Cas12a (LbCas12a)-4NLS
FEATURES             Location/Qualifiers
     source          1..8125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(761..1843)
                     /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     1805..1824
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1844..1921)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(2021..2026,2027..2042,2043..2049)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2055..2071
                     /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           2093..2226
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             2239..2256
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2668..2691
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2701..2721
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2731..2751
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6472..6492
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6502..6522
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6532..6552
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6562..6582
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     3'UTR           6593..6737
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     terminator      6813..6884
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        6990..7094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7095..7163,7164..7955)
                     /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(7313..7332)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatag tcatgccccg
      661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
      721 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
      781 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
      841 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
      901 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      961 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     1021 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     1081 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     1141 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     1201 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     1261 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     1321 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     1381 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     1441 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     1501 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     1561 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     1621 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     1681 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     1741 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     1801 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1861 gactctcttc cgggcactat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1921 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1981 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgtgg ttgacaatta atcatcggct
     2041 cgtataatgt gtaattgtga gcggataaca atttgatgtg ctagcgcata tccagtgtag
     2101 taaggcaagt cccttcaaga gttatcgttg atacccctcg tagtgcacat tcctttaacg
     2161 cttcaaaatc tgtaaagcac gccatatcgc cgaaaggcac acttaattat taagaggtaa
     2221 tacaccatga aatcttctca ccatcaccat caccatggtt ctagcaaaag caatgaaccg
     2281 ggtaaagcaa ccggtgaagg taaaccggtt aataacaaat ggctgaacaa tgccggtaaa
     2341 gatctgggta gtccggttcc ggatcgtatt gcaaataaac tgcgtgataa agaattcgag
     2401 agcttcgatg attttcgtga aaccttttgg gaagaagtta gcaaagatcc tgaactgagc
     2461 aaacagttta gccgcaataa caatgatcgt atgaaagttg gtaaagcacc gaaaacacgt
     2521 acccaggatg ttagcggtaa acgtacctca tttgaactga atcatcagaa accgattgaa
     2581 cagaatggtg gcgtttatga tatggataac attagcgttg ttaccccgaa acgcaacatt
     2641 gatattgaag gcggtggtgg tggttctttg gaggttttgt tccagggtcc aaacgcgaca
     2701 ccaaagaaaa agcgtaaagt cgggggatct ccgaaaaaga aacggaaagt aggtatccac
     2761 ggagtcccag cagctaccat gagcaaactg gagaagttca cgaactgtta ttctctgtcg
     2821 aaaaccttac gttttaaggc cattccggta ggtaaaacgc aagaaaatat cgacaataaa
     2881 cgtctcctgg ttgaagatga aaagcgtgcc gaagattata aaggtgttaa aaagctgctt
     2941 gatcgctatt acctgtcttt tatcaacgat gttctgcatt ccatcaaatt gaaaaacttg
     3001 aataattaca tctcgttatt tcgcaaaaaa acccgtaccg aaaaagaaaa caaggagctg
     3061 gaaaaccttg aaattaatct gcgtaaagaa atcgcgaaag catttaaagg taatgaagga
     3121 tataaatctc tgtttaaaaa agacattatt gaaaccatcc tgcctgagtt cttggatgat
     3181 aaagatgaaa ttgccctggt taatagcttt aatagattta cgactgcatt taccggcttt
     3241 tttgataacc gtgaaaacat gttcagcgaa gaagcgaaat cgacttccat tgccttccgt
     3301 tgcattaatg aaaacctgac cgtttacatt tctaatatgg acatctttga aaaagttgat
     3361 gccattttcg acaaacatga agttcaagaa atcaaagaaa agattttaaa ctccgattat
     3421 gatgttgaag atttctttga gggagaattt ttcaattttg ttctcaccca agagggtatt
     3481 gatgtctata acgccattat cggaggattt gttacggaat caggcgaaaa gattaaagga
     3541 ctgaatgaat acattaattt atacaatcag aaaaccaaac agaaattgcc aaaattcaaa
     3601 ccactgtata aacaggtatt aagcgaccgt gaatccttgt ccttttatgg cgaaggctat
     3661 acaagcgatg aagaggtgct cgaagtattc cgtaatactt tgaacaaaaa ctcggaaatc
     3721 ttctccagca ttaaaaaact ggaaaaattg tttaaaaatt tcgatgaata tagtagcgca
     3781 ggcatcttcg tcaaaaatgg ccctgcgatt tcaaccattt ctaaagacat tttcggtgaa
     3841 tggaacgtga tccgcgataa atggaacgcg gagtatgacg acattcatct gaaaaaaaaa
     3901 gccgttgtta ccgaaaagta tgaagatgac cgccgcaaga gctttaaaaa aattggtagt
     3961 ttctcactgg aacagttaca agaatatgca gacgccgatc tgtcggttgt ggaaaagtta
     4021 aaagagatta ttatccagaa agtcgatgaa atttataaag tgtacggctc cagcgaaaag
     4081 ctctttgatg ccgactttgt tttagaaaaa tcgctgaaaa aaaacgatgc cgtggtggcc
     4141 atcatgaaag atctgttgga ttcggtcaaa tcgttcgaaa actatatcaa ggcgttcttt
     4201 ggcgaaggga aagaaaccaa tcgtgacgaa tcgttctatg gcgattttgt gttggcgtat
     4261 gatattcttc tgaaagtaga tcacatctat gatgctatcc gcaactacgt gacccagaaa
     4321 ccgtattcga aggataaatt taagctgtat tttcaaaacc ctcagtttat gggtggctgg
     4381 ggtaaagata aagaaaccga ctaccgcgcg acgatcttgc gctatggctt taagtactat
     4441 ttagccatca tggataaaaa gtatgcaaaa tgcctccaga aaatcgataa ggatgatgtc
     4501 aacgggaact acgaaaaaat caactacaaa ctcttgcctg gcccgaataa aatgctgccg
     4561 aaagtctttt tctccaaaaa atggatggcc tattataacc cttccgaaga tattcagaag
     4621 atttacaaga acggtacctt taaaaaaggt gacatgttta atctgaatga ctgtcacaag
     4681 cttattgatt tcttcaaaga tagtatttct cgctacccta aatggtcaaa cgcgtacgat
     4741 tttaatttct cagaaacaga gaaatacaaa aatattgcgg ggttttatcg cgaagtcgaa
     4801 gaacaagggt ataaggtgtc gttcgaatcg gcttccaaaa aggaagtgga taaacttgtt
     4861 gaagaaggca aactctatat gttccagatc tacaacaaag atttctccga caagagccat
     4921 ggcacgccga acttacatac catgtacttt aaattactgt tcgatgaaaa taatcatggt
     4981 cagattcgct tgagcggtgg cgccgaactg tttatgcgcc gcgcatcatt gaaaaaggaa
     5041 gaacttgttg tgcacccagc aaacagtccg attgcaaaca aaaatccgga taacccgaag
     5101 aaaactacca ccttaagcta tgatgtatat aaagataaac gttttagcga agatcagtat
     5161 caactccaca ttccgatcgc gattaacaaa tgtcctaaga acatctttaa aatcaatacc
     5221 gaggttcgtg tactgctcaa acacgacgac aacccctatg ttattggcat tgaccgcggc
     5281 gaacgcaacc tgctgtatat tgtcgttgtg gatggcaaag gtaacattgt tgaacagtat
     5341 agccttaatg aaatcatcaa taatttcaac ggcattcgta ttaaaacgga ctatcatagc
     5401 ctgctggaca aaaaagagaa agaacgcttc gaagcccgtc agaattggac ctctatcgaa
     5461 aatatcaagg aactgaaagc tgggtacatt tctcaagtgg tccacaagat ttgcgaactg
     5521 gtggaaaagt atgacgcggt gatcgcactg gaggatctga attcgggctt taaaaatagc
     5581 cgtgtgaagg tggagaaaca ggtttaccaa aagttcgaaa aaatgctgat tgacaaactt
     5641 aattacatgg tggacaaaaa atctaatcca tgtgcgaccg gcggggctct caaaggctat
     5701 cagattacga ataaattcga aagcttcaaa tccatgtcta cccagaacgg atttatcttt
     5761 tacatccccg cctggctgac ttccaaaatt gatccgagta ccggttttgt taatctgctg
     5821 aaaaccaaat atacgtcgat cgccgattca aaaaaattca ttagcagctt cgaccgtatt
     5881 atgtacgtcc cggaagaaga cctgttcgaa ttcgcccttg actataaaaa tttctcgcgt
     5941 acggatgccg attatatcaa aaaatggaaa ctgtacagct acggcaaccg catccgcatt
     6001 tttcgcaacc caaagaagaa caacgtattt gactgggagg aagtttgctt gacgagcgct
     6061 tacaaagagc tctttaacaa atatgggatc aattatcagc aaggggacat ccgtgcgttg
     6121 ctgtgtgagc agtcagataa agcgttctac agctctttca tggctttaat gtcgctgatg
     6181 ttgcagatgc gcaattcgat cactggccgc accgatgtgg atttcctgat tagcccggtc
     6241 aaaaatagcg atggcatttt ttacgattca cgtaactatg aagcccagga aaacgctatt
     6301 ctgccgaaga acgctgatgc gaacggcgcg tacaatattg cccgtaaggt actgtgggca
     6361 attggtcagt tcaagaaagc ggaagatgaa aagttggata aggtcaaaat tgcgatctcg
     6421 aataaagaat ggctggaata tgcgcagacc agcgtgaaac atggcggatc accgaagaag
     6481 aaacgcaaag tcggaggtag ccctaagaag aagcgtaagg ttggggggtc gccaaagaag
     6541 aaacgtaaag ttgggggctc gccgaaaaag aaacgcaagg tgtaataggt aatctctgct
     6601 taaaagcaca gaatctaaga tccctgccat ttggcgggga tttttttatt tgttttcagg
     6661 aaataaataa tcgatcgcgt aataaaatct attattattt ttgtgaagaa taaatttggg
     6721 tgcaatgaga atgcgcaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     6781 ctgacacatg tcgagtaagg atctccaggc atcaaataaa acgaaaggct cagtcgaaag
     6841 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcgtcgac actatggttg
     6901 ctttgacgta tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     6961 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     7021 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     7081 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     7141 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     7201 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     7261 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     7321 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     7381 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     7441 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     7501 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     7561 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     7621 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     7681 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     7741 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     7801 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     7861 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     7921 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     7981 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     8041 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     8101 ccgtagaaaa gatcaaagga tcttc
//
