LOCUS       sequence-220502-        8762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter expression plasmid for human codon optimized Mb3Cas12a 
            nuclease with C-terminal NLS and HA tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8762)
  AUTHORS   Marino ND, Zhang JY, Borges AL, Sousa AA, Leon LM, Rauch BJ, Walton 
            RT, Berry JD, Joung JK, Kleinstiver BP, Bondy-Denomy J
  TITLE     Discovery of widespread Type I and Type V CRISPR-Cas inhibitors.
  JOURNAL   Science. 2018 Sep 6. pii: science.aau5174. doi: 
            10.1126/science.aau5174.
  PUBMED    30190308
REFERENCE   2  (bases 1 to 8762)
  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: "Science. 
            2018 Sep 6. pii: science.aau5174. doi: 10.1126/science.aau5174."
COMMENT     Sequence Label: pCAG-hMb3Cas12a-NLS(nucleoplasmin)-3xHA (RTW2500)
FEATURES             Location/Qualifiers
     source          1..8762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             5560..5607
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5614..5694
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(5784..5803)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5849..5904
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5903..5922)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6265..6281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6265..6281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6278..6300)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6289..6305
                     /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)."
     promoter        complement(join(6313..6319,6320..6337,6338..6343))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6358..6379
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6438..6633
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6484..6619
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6639..6773
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6676..6695)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6730..6749
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6841..6858)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7012..7600)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7092..7111)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7771..8562,8563..8631))
                     /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     8394..8413
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8632..8736)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggsgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tctgcagtcg
     1741 acggtaccgc gggcccggga tccaccggtc gccaccatgc tgttccagga cttcacccac
     1801 ctgtaccccc tgagcaagac cgtcagattc gagctgaagc ccatcggcaa gaccctggaa
     1861 cacatccacg ccaagaactt tctgaaccag gacgagacaa tggccgacat gtaccagaaa
     1921 gtgaaggcca tcctggacga ctaccaccgg gactttatcg ccgacatgat gggcgaagtg
     1981 aagctgacca agctggccga gttctacgac gtgtacctga agttccggaa gaaccccaag
     2041 gacgacggcc tgcagaagca gctgaaagac ctgcaggctg tgctgcggaa agaaatcgtg
     2101 aagcctatcg gaaacggcgg caagtacaag gccggctacg acagactgtt cggcgccaag
     2161 ctgttcaagg acggcaaaga gctgggcgac ctggccaagt tcgtgatcgc ccaggaaggc
     2221 gagagcagcc ctaagctggc ccacctggcc catttcgaga agttcagcac ctacttcacc
     2281 ggcttccacg acaaccgcaa gaacatgtac agcgacgagg acaagcacac cgccattgcc
     2341 taccggctga tccacgagaa cctgccccgg ttcatcgaca acctgcagat cctggccacc
     2401 atcaagcaga agcacagcgc cctgtacgac cagatcatca acgagctgac cgccagcggc
     2461 ctggatgtgt ctctggcctc tcacctggat ggctaccaca agctgctgac acaggaaggc
     2521 atcaccgcct acaacaccct gctgggcgga atctctggcg aggccggcag cagaaagatc
     2581 cagggcatca atgaactgat caacagccac cacaaccagc actgccacaa gagcgagcgg
     2641 attgccaagc tgcggcccct gcacaagcag attctgagcg acggaatggg cgtgtccttc
     2701 ctgcccagca agttcgccga cgacagcgaa gtgtgccagg ccgtgaatga gttctaccgg
     2761 cactacgccg acgtgttcgc caaggtgcag agcctgttcg acggcttcga cgactatcag
     2821 aaagacggca tctacgtgga gtacaagaac ctgaacgagc tgtccaagca ggccttcggc
     2881 gacttcgctc tgctgggaag agtgctggac ggctactatg tggacgtcgt gaaccccgag
     2941 ttcaacgaga gatttgccaa ggccaagacc gacaacgcca aagccaagct gacaaaagag
     3001 aaggacaagt tcatcaaggg cgtgcactcc ctggcttctc tggaacaggc catcgagcac
     3061 tacaccgcca gacacgacga cgagtctgtg caggccggaa agctgggcca gtacttcaag
     3121 cacggactgg ccggcgtgga caaccccatc cagaagatcc acaacaacca ctctacaatc
     3181 aagggcttcc tggaaagaga gcggcctgcc ggcgaaagag ccctgcccaa gatcaagagc
     3241 gacaagagcc ccgagatcag acagctgaaa gagctgctgg acaacgccct gaacgtggcc
     3301 cacttcgcca aactgctgac caccaagacc acactgcaca atcaggatgg caacttctac
     3361 ggcgagttcg gagccctgta tgatgagctg gccaagatcg ccaccctgta caacaaagtg
     3421 cgggactacc tgagccagaa gcccttcagc accgagaagt acaagctgaa tttcggcaac
     3481 cctaccctgc tgaacggctg ggacctgaac aaagagaaag ataacttcgg cgtgatcctg
     3541 cagaaggacg gctgctacta cctggccctg ctggataagg cccacaagaa ggtgttcgat
     3601 aacgccccca acaccggcaa gtctgtgtat cagaagatga tctataagct gctgcctggc
     3661 cccaacaaga tgctgcccaa ggtgttcttc gccaagagca acctggacta ctacaacccc
     3721 agcgccgaac tgctggacaa atacgcccag ggcacacaca agaaaggcga caacttcaac
     3781 ctgaaggact gccacgccct gatcgatttc ttcaaggccg gaatcaacaa gcaccccgag
     3841 tggcagcact tcggcttcaa gttcagcccc accagcagct accaggacct gagcgacttc
     3901 tacagagagg tggaacccca gggctaccaa gtgaagttcg tggacatcaa cgccgactac
     3961 atcaatgagc tggtggaaca gggccagctg tacctgtttc agatctacaa caaggacttt
     4021 agccccaagg cccacggcaa gcccaacctg cacaccctgt atttcaaggc cctgtttagc
     4081 gaggacaacc tcgtgaatcc catctacaaa ctgaacgggg aggccgagat cttctaccgg
     4141 aaggcctccc tggacatgaa cgagacaacc atccacagag ccggcgaggt gctggaaaac
     4201 aagaaccctg acaaccctaa gaaacggcag ttcgtgtacg acatcatcaa ggacaagcgg
     4261 tacacccagg ataagttcat gctgcacgtg cccatcacca tgaacttcgg agtgcagggc
     4321 atgaccatca aagagttcaa caaaaaagtg aaccagagca tccagcagta cgacgaagtg
     4381 aacgtgatcg gcatcgaccg gggcgagcgg catctgctgt atctgaccgt gatcaactcc
     4441 aagggcgaga ttctggaaca gagatccctg aacgacatca ccaccgcctc cgccaacggc
     4501 acccagatga ccacccctta ccacaagatc ctggacaagc gcgagatcga gcggctgaac
     4561 gccagagtgg gatggggaga gatcgagaca atcaaagaac tgaagtccgg ctacctgtcc
     4621 cacgtggtgc atcagatctc ccagctgatg ctgaagtaca acgccatcgt ggtgctggaa
     4681 gatctgaatt ttggcttcaa gaggggccgg ttcaaggtgg aaaagcagat ctaccagaac
     4741 ttcgagaatg ccctgatcaa gaaactgaac cacctggtgc tgaaagacaa ggccgacgac
     4801 gagatcggca gctacaagaa cgccctgcag ctgactaaca acttcaccga tctgaagagt
     4861 atcggcaagc agaccggctt tctgttctac gtgcccgcct ggaataccag caagatcgac
     4921 cccgagacag gcttcgtgga cctgctgaag cctagatacg agaatatcgc ccagagccag
     4981 gccttcttcg gcaagttcga caagatctgc tacaacgccg ataggggcta cttcgagttc
     5041 cacatcgact acgccaagtt caatgacaag gccaaaaaca gccggcagat ctggaaaatc
     5101 tgcagccacg gcgataagcg ctacgtgtac gataagaccg ccaaccagaa caagggcgcc
     5161 accatcggag tgaatgtgaa cgatgagctg aagtccctgt tcacccgcta ccacatcaac
     5221 gataagcagc ccaatctcgt gatggacatc tgccagaaca acgacaaaga gtttcacaag
     5281 agcctgatgt atctgctgaa aacactgctg gctctgcggt acagcaacgc cagctccgac
     5341 gaggatttca tcctgagccc cgtggccaac gacgagggcg tgttcttcaa tagcgccctg
     5401 gccgacgata cccagcccca gaatgccgat gccaacggcg cctaccacat tgccctgaag
     5461 ggactgtggc tgctgaacga actgaagaac agcgacgatc tgaacaaagt gaagctggcc
     5521 atcgacaacc agacctggct gaatttcgcc cagaacagaa agaggccggc ggccacgaag
     5581 aaggccggcc aggccaagaa gaagaaggga tcctacccat acgatgttcc agattacgct
     5641 tatccctacg acgtgcctga ttatgcatac ccatatgatg tccccgacta tgcctaaagc
     5701 ggccgcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     5761 ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg
     5821 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     5881 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     5941 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     6001 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     6061 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     6121 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     6181 ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg
     6241 cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     6301 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6361 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6421 gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     6481 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     6541 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     6601 ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata
     6661 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     6721 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca
     6781 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     6841 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     6901 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     6961 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     7021 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     7081 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     7141 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     7201 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     7261 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     7321 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     7381 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     7441 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7501 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     7561 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     7621 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     7681 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     7741 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     7801 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     7861 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     7921 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     7981 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     8041 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     8101 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     8161 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     8221 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     8281 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     8341 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     8401 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     8461 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     8521 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     8581 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     8641 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     8701 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     8761 tg
//
