LOCUS       sequence-486081-        8080 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Bacterial expression of LbCas12a-RVQ 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 8080)
  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 8080)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 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: pEM6_CL7_Bac_2NLS-RVQ (LbCas12a)-2NLS
FEATURES             Location/Qualifiers
     source          1..8080
                     /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             2635..2658
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2668..2688
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2698..2718
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6436..6456
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6466..6486
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6490..6513
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6520..6537
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     3'UTR           6548..6692
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     terminator      6768..6839
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        6945..7049
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7050..7118,7119..7910)
                     /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(7268..7287)
                     /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 cgggcgctat 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 gcaaaagcaa tgaaccgggt aaagcaaccg gtgaaggtaa accggttaat
     2281 aacaaatggc tgaacaatgc cggtaaagat ctgggtagtc cggttccgga tcgtattgca
     2341 aataaactgc gtgataaaga attcgagagc ttcgatgatt ttcgtgaaac cttttgggaa
     2401 gaagttagca aagatcctga actgagcaaa cagtttagcc gcaataacaa tgatcgtatg
     2461 aaagttggta aagcaccgaa aacacgtacc caggatgtta gcggtaaacg tacctcattt
     2521 gaactgaatc atcagaaacc gattgaacag aatggtggcg tttatgatat ggataacatt
     2581 agcgttgtta ccccgaaacg caacattgat attgaaggcg gtggtggtgg ttctttggag
     2641 gttttgttcc agggtccaaa cgcgacacca aagaaaaagc gtaaagtcgg gggatctccg
     2701 aaaaagaaac ggaaagtagg tatccacgga gtcccagcag ctaccatgag caaactggag
     2761 aagttcacga actgttattc tctgtcgaaa accttacgtt ttaaggccat tccggtaggt
     2821 aaaacgcaag aaaatatcga caataaacgt ctcctggttg aagatgaaaa gcgtgccgaa
     2881 gattataaag gtgttaaaaa gctgcttgat cgctattacc tgtcttttat caacgatgtt
     2941 ctgcattcca tcaaattgaa aaacttgaat aattacatct cgttatttcg caaaaaaacc
     3001 cgtaccgaaa aagaaaacaa ggagctggaa aaccttgaaa ttaatctgcg taaagaaatc
     3061 gcgaaagcat ttaaaggtaa tgaaggatat aaatctctgt ttaaaaaaga cattattgaa
     3121 accatcctgc ctgagttctt ggatgataaa gatgaaattg ccctggttaa tagctttaat
     3181 agatttacga ctgcatttac cggctttttt gataaccgtg aaaacatgtt cagcgaagaa
     3241 gcgaaatcga cttccattgc cttccgttgc attaatgaaa acctgaccgt ttacatttct
     3301 aatatggaca tctttgaaaa agttgatgcc attttcgaca aacatgaagt tcaagaaatc
     3361 aaagaaaaga ttttaaactc cgattatgat gttgaagatt tctttgaggg agaatttttc
     3421 aattttgttc tcacccaaga gggtattgat gtctataacg ccattatcgg aggatttgtt
     3481 acggaatcag gcgaaaagat taaaggactg aatgaataca ttaatttata caatcagaaa
     3541 accaaacaga aattgccaaa attcaaacca ctgtataaac aggtattaag cgaccgtgaa
     3601 tccttgtcct tttatggcga aggctataca agcgatgaag aggtgctcga agtattccgt
     3661 aatactttga acaaaaactc ggaaatcttc tccagcatta aaaaactgga aaaattgttt
     3721 aaaaatttcg atgaatatag tagcgcaggc atcttcgtca aaaatggccc tgcgatttca
     3781 accatttcta aagacatttt cggtgaatgg aacgtgatcc gcgataaatg gaacgcggag
     3841 tatgacgaca ttcatctgaa aaaaaaagcc gttgttaccg aaaagtatga agatgaccgc
     3901 cgcaagagct ttaaaaaaat tggtagtttc tcactggaac agttacaaga atatgcagac
     3961 gccgatctgt cggttgtgga aaagttaaaa gagattatta tccagaaagt cgatgaaatt
     4021 tataaagtgt acggctccag cgaaaagctc tttgatgccg actttgtttt agaaaaatcg
     4081 ctgaaaaaaa acgatgccgt ggtggccatc atgaaagatc tgttggattc ggtcaaatcg
     4141 ttcgaaaact atatcaaggc gttctttggc gaagggaaag aaaccaatcg tgacgaatcg
     4201 ttctatggcg attttgtgtt ggcgtatgat attcttctga aagtagatca catctatgat
     4261 gctatccgca actacgtgac ccagaaaccg tattcgaagg ataaatttaa gctgtatttt
     4321 caaaaccctc agtttatggg tggctgggat aaagataaag aaaccgacta ccgcgcgacg
     4381 atcttgcgct atggctctaa gtactattta gccatcatgg ataaaaagta tgcaaaatgc
     4441 ctccagaaaa tcgataagga tgatgtcaac gggaactacg aaaaaatcaa ctacaaactc
     4501 ttgcctggcc cgaataaaat gctgccgaaa gtcttttttt ccaaaaaatg gatggcctat
     4561 tataaccctt ccgaagatat tcagaagatt tacaagaacg gtacctttaa aaaaggtgac
     4621 atgtttaatc tgaatgactg tcacaagctt attgatttct tcaaagatag tatttctcgc
     4681 taccctaaat ggtcaaacgc gtacgatttt aatttctcag aaacagagaa atacaaagat
     4741 attgcggggt tttatcgcga agtcgaagaa caagggtata aggtgtcgtt cgaatcggct
     4801 tccaaaaagg aagtggataa acttgttgaa gaaggcaaac tctatatgtt ccagatctac
     4861 aacaaagatt tctccgacaa gagccatggc acgccgaact tacataccat gtactttaaa
     4921 ttactgttcg atgaaaataa tcatggtcag attcgcttga gcggtggcgc cgaactgttt
     4981 atgcgccgcg catcattgaa aaaggaagaa cttgttgtgc acccagcaaa cagtccgatt
     5041 gcaaacaaaa atccggataa cccgaagaaa actaccacct taagctatga tgtatataaa
     5101 gataaacgtt ttagcgaaga tcagtatcaa ctccacattc cgatcgcgat taacaaatgt
     5161 cctaagaaca tctttaaaat caataccgag gttcgtgtac tgctcaaaca cgacgacaac
     5221 ccctatgtta ttggcattga ccgcggcgaa cgcaacctgc tgtatattgt cgttgtggat
     5281 ggcaaaggta acattgttga acagtatagc cttaatgaaa tcatcaataa tttcaacggc
     5341 attcgtatta aaacggacta tcatagcctg ctggacaaaa aagagaaaga acgcttcgaa
     5401 gcccgtcaga attggacctc tatcgaaaat atcaaggaac tgaaagctgg gtacatttct
     5461 caagtggtcc acaagatttg cgaactggtg gaaaagtatg acgcggtgat cgcactggag
     5521 gatctgaatt cgggctttaa aaatagccgt gtgaaggtgg agaaacaggt ttaccaaaag
     5581 ttcgaaaaaa tgctgattga caaacttaat tacatggtgg acaaaaaatc taatccatgt
     5641 gcgaccggcg gggctctcaa aggctatcag attacgaata aattcgaaag cttcaaatcc
     5701 atgtctaccc agaacggatt tatcttttac atccccgcct ggctgacttc caaaattgat
     5761 ccgagtaccg gttttgttaa tctgctgaaa accaaatata cgtcgatcgc cgattcaaaa
     5821 aaattcatta gcagcttcga ccgtattatg tacgtcccgg aagaagatct gttcgaattc
     5881 gcccttgact ataaaaattt ctcgcgtacg gatgccgatt atatcaaaaa atggaaactg
     5941 tacagctacg gcaaccgcat ccgcattttt cgcaacccaa agaagaacaa cgtatttgac
     6001 tgggaggaag tttgcttgac gagcgcttac aaagagctct ttaacaaata tgggatcaat
     6061 tatcagcaag gggacatccg tgcgttgctg tgtgagcagt cagataaagc gttctacagc
     6121 tctttcatgg ctttaatgtc gctgatgttg cagatgcgca attcgatcac tggccgcacc
     6181 gatgtggatt tcctgattag cccggtcaaa aatagcgatg gcatttttta cgattcacgt
     6241 aactatgaag cccaggaaaa cgctattctg ccgaagaacg ctgatgcgaa cggcgcgtac
     6301 aatattgccc gtaaggtact gtgggcaatt ggtcagttca agaaagcgga agatgaaaag
     6361 ttggataagg tcaaaattgc gatctcgaat aaagaatggc tggaatatgc gcagaccagc
     6421 gtgaaacatg ggtcacctaa aaaaaagaga aaagtaggcg gatctcctaa gaagaagaga
     6481 aaagtttcat tagaagtgct ttttcagggc ccaggttctc accatcacca tcaccattaa
     6541 taggtaatct ctgcttaaaa gcacagaatc taagatccct gccatttggc ggggattttt
     6601 ttatttgttt tcaggaaata aataatcgat cgcgtaataa aatctattat tatttttgtg
     6661 aagaataaat ttgggtgcaa tgagaatgcg caggcccttt cgtctcgcgc gtttcggtga
     6721 tgacggtgaa aacctctgac acatgtcgag taaggatctc caggcatcaa ataaaacgaa
     6781 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcg
     6841 tcgacactat ggttgctttg acgtatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     6901 aaataccgca tcaggcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     6961 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     7021 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     7081 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     7141 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     7201 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     7261 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     7321 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     7381 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     7441 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     7501 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     7561 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     7621 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     7681 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     7741 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     7801 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     7861 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     7921 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     7981 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     8041 actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
