LOCUS       sequence-383108-        9323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  vector for encoding a human codon-optimized Cas12Max driven by CBh 
            promoter, guide RNAs compatible with xCas12i driven by hU6, and 
            mCherry driven by CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9323)
  AUTHORS   Zhang H, Kong X, Xue M, Hu J, Wang Z, Wei Y, Wang H, Zhou J, Zhang 
            W, Xu M, Shen X, Yin F, Ai Z, Huang G, Xia J, Song X, Li H, Yuan Y, 
            Li J, Zhong N, Zhang M, Zhou Y, Yang H
  TITLE     An engineered xCas12i with high activity, high specificity, and 
            broad PAM range.
  JOURNAL   Protein Cell. 2023 Jun 28;14(7):538-543. doi: 
            10.1093/procel/pwac052.
  PUBMED    37378659
REFERENCE   2  (bases 1 to 9323)
  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"; doi: "10"; journalName: 
            "Protein Cell"; date: "2023-06-28- 28"; volume: "14"; issue: "7"; 
            pages: "538-543"
COMMENT     Sequence Label: Cas12Max
FEATURES             Location/Qualifiers
     source          1..9323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        301..376
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        473..758
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        760..1037
                     /label=chicken beta-actin promoter
     intron          1038..1265
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1277..1286
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1286..1351
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1358..1378
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4643..4690
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4718..4735)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4724..4931
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        5000..5303
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5304..5507
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5458..5478
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             5536..6246
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5681..5699)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     rep_origin      6801..7256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6888..6907)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7098..7119
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7273..7292)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7392..7414
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7452..7470)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7538..7642
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7643..7711,7712..8503)
                     /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(7861..7880)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8674..9262
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9163..9182
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg agaaatgtgt ccccagttga cacccattac agtaggagca tacttttttt
      301 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
      361 ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag
      421 tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagaggta cccgttacat
      481 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      541 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      601 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      661 acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt
      721 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
      781 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
      841 aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg
      901 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
      961 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     1021 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     1081 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     1141 gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat
     1201 ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt
     1261 ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact acaaggatca
     1321 tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg
     1381 tatccacgga gtcccagcag ccatgagttc tgatgtggtg cggccttata acacaaagct
     1441 gctcccagat aacagaaagc acaatatgtt cctgcagacc ttcaagcggc tgaacagcat
     1501 ctctctgaac cacttcgacc tgctgatctg cctgtacgct gcaatcacca acaagaaggc
     1561 cgaggaatac aagtctgaaa aggaagccca cgtgaccgcc gatagcctgt gtgccatcaa
     1621 ttggttcaga cccatgagca agagatacag caaatacgcc accaccacct tcaacatgtt
     1681 agaactgttt aaggagtaca gcggccacga gcctgatgcc tattccaaga actacctgat
     1741 gagcaatatc gacagcgaca gattcgtgtg ggtggattgt aggaagttcg ctaaggactt
     1801 tgcctatcag atggaactgg gtttccacga gttcaccgtg ttggccgaaa ccctgctggc
     1861 taattctatc ctggtgctga acgagagcac caaggccaat tgggcttggg gaaccgtgtc
     1921 tgccctgtac ggcggcggag ataaggagga cagcacactg aagagcaaga ttctgctggc
     1981 cttcgtggac gccctgaaca accacgagct gaaaacaaag agagaaatct tgaatcaagt
     2041 gtgtgaatct ctgaaatacc agagctacca ggacatgtac gtggatttta gaagcgtggt
     2101 tgacgaaaac ggcaacaaga agtctcctag aggctctatg cctatcgtga ccaagttcga
     2161 gacagacgac ctgatcagcg acaaccaaag aaaggccatg atcagcaact tcactaagaa
     2221 cgccgctgcc aaggcagcta agaaacctat cccttacttg gaccgcctga aggagcacat
     2281 ggtgtccctg tgcgacgagt acaatgtgta tgcctgggcc gcggccatca caaacagcaa
     2341 cgccgacgtg accgcccgga ataccagaaa cctgacattc atcggcgaac agaacagcag
     2401 acgaaaggaa ctgagcgtgc tgcagacaac aaccaacgag aaggctaagg acatcctgaa
     2461 caagatcaac gacaacctga ttcaggaggt gcggtacacc cctgccccta agcacctggg
     2521 cagagatctg gccaacctgt ttgatacact gaaggaaaag gacatcaaca acatcgagaa
     2581 cgaagaagag aaacagaacg tgatcaatga ctgtatcgag cagtacgtgg acgattgcag
     2641 aagcctcaac cggaacccca tcgcagccct cctgaagcac atctctaggt actacgagga
     2701 tttcagcgcc aagaatttcc tggacggcgc caagctgaac gtgctgactg aggtggtgaa
     2761 ccggcagaag gcccacccca ccatctggag cgagaaggct tacacctgga tcagcaagtt
     2821 cgacaagaac cggagacagg ccaacagcag cctggtcgga tgggttgtgc cccccgagga
     2881 ggtgcacaag gagaaaatcg ccggacagca gagcatgatg tgggtgaccc tcaccctgct
     2941 ggacgacggc aagtgggtca aacatcacat ccccttcagc gacagcagat actacagcga
     3001 agtgtacgcc tacaacccta atctgcctta tctggacgga ggcatcccaa gacagagcaa
     3061 gttcggcaac aaaccaacaa ccaacctgac agccgagtcc caggccctcc tggctaattc
     3121 taagtacaag aaagccaaca agagcttcct gcgggctaaa gagaatgcca cacacaacgt
     3181 gcgggtgtcc cctaacacct ctctgtgcat tagactgctg aaggacagcg ccggaaacca
     3241 gatgttcgac aaaatcggca acgtgctctt cggcatgcag atcaaccaca agatcaccgt
     3301 gggaaaacct aactacaaga tcgaggtggg cgacagattc ctgggcttcg atcagaacca
     3361 gagcgagaac cacacctacg ccgtgctgca gagagtgtcc gagagcagtc acgacaccca
     3421 ccactttaac ggctgggacg tgaaggtgct ggaaaagggc aaagtgacca gcgatgtgat
     3481 cgtgcgggac gaggtctacg accaactgtc ttacgagggc gtcccctacg atagcagcaa
     3541 gttcgccgag tggcgggaca agcgcagaag atttgtgctt gagaacctga gcatccagct
     3601 ggaagagggc aagaccttcc tgacagagtt cgacaagctg aataaggaca gcctgtaccg
     3661 ctggaacatg aactacctga aactgctgag aaaggccatc cgggccggag gcaaagagtt
     3721 cgccaagatc gctaagacag agatcttcga gctggcggtg gaaagattcg gccctattaa
     3781 cctgggcagc ctgtcccaga tcagccttaa gatgattgcc tcctttaagg gcgtggtcca
     3841 gtcctacttc tccgtgagcg gctgcgtgga tgatgcctcc aaaaaggccc atgattctat
     3901 gctgttcaca tttatgtgcg ccgccgaaga aaagcggacc aacaagagag aagaaaagac
     3961 caacagagcc gccagcttta tcctgcaaaa agcctacctg catggctgca agatgatcgt
     4021 gtgcgaggac gaccttcctg tggccgacgg caagacaggc aaagcccaga atgccgaccg
     4081 gatggactgg tgcgccagag ccctggccaa gaaggtgaac gacggctgtg ttgccatgag
     4141 catctgctac agagctatcc ctgcctacat gagcagccac caggacccct ttgtgcacat
     4201 gcaggataag aaaaccagcg tgctgcggcc tagattcatg gaagttaata aggatagcat
     4261 cagagactac cacgtggcgg gcctgagaag aatgctgaac agcaagagtg acgctggcac
     4321 cagtgtttat taccggcaag ctgccctgca tttctgcgaa gccctgggcg tgagccctga
     4381 actggtgaaa aacaagaaaa cccacgccgc cgaactgggc aagcacatgg gcagcgctat
     4441 gctgatgccc tggagaggcg gtagagtgta catcgccagc aaaaagctga cctccgatgc
     4501 caaatcagtg aagtactgcg gcgaggatat gtggcagtac cacgccgatg agatcgccgc
     4561 tgttaacatc gccatgtatg aggtgtgctg ccagaccggc gctttcggaa agaaacagaa
     4621 aaaatcggac gagctgcctg gaaaaaggcc ggcggccacg aagaaggccg gccaggcaaa
     4681 gaagaagaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     4741 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4801 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4861 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg
     4921 catgctgggg agcggccgct agttattaat agtaatcaat tacggggtca ttagttcata
     4981 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     5041 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     5101 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     5161 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     5221 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     5281 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     5341 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     5401 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     5461 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
     5521 gtcagatccg ctagcatggt gagcaagggc gaggaggata acatggccat catcaaggag
     5581 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
     5641 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     5701 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     5761 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     5821 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     5881 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     5941 tccgacggcc ccgtaatgca gaagaaaacc atgggctggg aggcctcctc cgagcggatg
     6001 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     6061 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     6121 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     6181 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     6241 aagtaaggat ccgggatgca gaaattgatg atctattaaa caataaagat gtccactaaa
     6301 atggaagttt ttcctgtcat actttgttaa gaagggtgag aacagagtac ctacattttg
     6361 aatggaagga ttggagctac gggggtgggg gtggggtggg attagataaa tgcctgctct
     6421 ttactgaagg ctctttacta ttgctttatg ataatgtttc atagttggat atcataattt
     6481 aaacaagcaa aaccaaatta agggccagct cattcctccc actcatgatc tatagatcta
     6541 tagatctctc gtgggatcat tgtttttctc ttgattccca ctttgtggtt ctaagtactg
     6601 tggtttccaa atgtgtcagt ttcatagcct gaagaacgag atcagcagcc tctgttccac
     6661 atacacttca ttctcagtat tgttttgcca agttctaatt ccatcagaag ctggtcgacc
     6721 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     6781 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     6841 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     6901 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     6961 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     7021 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     7081 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     7141 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     7201 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     7261 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     7321 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     7381 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     7441 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     7501 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     7561 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     7621 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     7681 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     7741 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     7801 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     7861 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     7921 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     7981 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     8041 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     8101 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     8161 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     8221 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     8281 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     8341 tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc
     8401 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     8461 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     8521 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     8581 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     8641 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     8701 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     8761 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     8821 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     8881 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     8941 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     9001 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     9061 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     9121 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     9181 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     9241 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     9301 ttgctggcct tttgctcaca tgt
//
