LOCUS       sequence-389250-        9321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  vector for encoding a human codon-optimized hfCas12Max 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 9321)
  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 9321)
  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: hfCas12Max
FEATURES             Location/Qualifiers
     source          1..9321
                     /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        299..374
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        471..756
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        758..1035
                     /label=chicken beta-actin promoter
     intron          1036..1263
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1275..1284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1284..1349
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1356..1376
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4641..4688
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4716..4733)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4722..4929
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        4998..5301
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5302..5505
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5456..5476
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             5534..6244
                     /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(5679..5697)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     rep_origin      6799..7254
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6886..6905)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7096..7117
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7271..7290)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7390..7412
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7450..7468)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7536..7640
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7641..7709,7710..8501)
                     /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(7859..7878)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8672..9260
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9161..9180
                     /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 cacaggtctt cgagaagacc ttttttttgt
      301 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      361 caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc
      421 cgtttttagc gcgtgcgcca attctgcaga caaatggctc tagaggtacc cgttacataa
      481 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      541 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      601 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      661 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
      721 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      781 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      841 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg
      901 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
      961 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
     1021 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
     1081 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
     1141 gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt taagggatgg
     1201 ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat cacttttttt
     1261 caggttggac cggtgccacc atggactata aggaccacga cggagactac aaggatcatg
     1321 atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta
     1381 tccacggagt cccagcagcc atgagttctg atgtggtgcg gccttataac acaaagctgc
     1441 tcccagataa cagaaagcac aatatgttcc tgcagacctt caagcggctg aacagcatct
     1501 ctctgaacca cttcgacctg ctgatctgcc tgtacgctgc aatcaccaac aagaaggccg
     1561 aggaatacaa gtctgaaaag gaagcccacg tgaccgccga tagcctgtgt gccatcaatt
     1621 ggttcagacc catgagcaag agatacagca aatacgccac caccaccttc aacatgttag
     1681 aactgtttaa ggagtacagc ggccacgagc ctgatgccta ttccaagaac tacctgatga
     1741 gcaatatcga cagcgacaga ttcgtgtggg tggattgtag gaagttcgct aaggactttg
     1801 cctatcagat ggaactgggt ttccacgagt tcaccgtgtt ggccgaaacc ctgctggcta
     1861 attctatcct ggtgctgaac gagagcacca aggccaattg ggcttgggga accgtgtctg
     1921 ccctgtacgg cggcggagat aaggaggaca gcacactgaa gagcaagatt ctgctggcct
     1981 tcgtggacgc cctgaacaac cacgagctga aaacaaagag agaaatcttg aatcaagtgt
     2041 gtgaatctct gaaataccag agctaccagg acatgtacgt ggattttaga agcgtggttg
     2101 acgaaaacgg caacaagaag tctcctagag gctctatgcc tatcgtgacc aagttcgaga
     2161 cagacgacct gatcagcgac aaccaaagaa aggccatgat cagcaacttc actaagaacg
     2221 ccgctgccaa ggcagctaag aaacctatcc cttacttgga ccgcctgaag gagcacatgg
     2281 tgtccctgtg cgacgagtac aatgtgtatg cctgggccgc ggccatcaca aacagcaacg
     2341 ccgacgtgac cgcccggaat accagaaacc tgacattcat cggcgaacag aacagcagac
     2401 gaaagagact gagcgtgctg cagacaacaa ccaacgagaa ggctaaggac atcctgaaca
     2461 agatcaacga caacctgatt caggaggtgc ggtacacccc tgcccctaag cacctgggca
     2521 gagatctggc caacctgttt gatacactga aggaaaagga catcaacaac atcgagaacg
     2581 aagaagagaa acagaacgtg atcaatgact gtatcgagca gtacgtggac gattgcagaa
     2641 gcctcaaccg gaaccccatc gcagccctcc tgaagcacat ctctaggtac tacgaggatt
     2701 tcagcgccaa gaatttcctg gacggcgcca agctgaacgt gctgactgag gtggtgaacc
     2761 ggcagaaggc ccaccccacc atctggagcg agaaggctta cacctggatc agcaagttcg
     2821 acaagaaccg gagacaggcc aacagcagcc tggtcggatg ggttgtgccc cccgaggagg
     2881 tgcacaagga gaaaatcgcc ggacagcaga gcatgatgtg ggtgaccctc accctgctgg
     2941 acgacggcaa gtgggtcaaa catcacatcc ccttcagcga cagcagatac tacagcgaag
     3001 tgtacgccta caaccctaat ctgccttatc tggacggagg catcccaaga cagagcaagt
     3061 tcggcaacaa accaacaacc aacctgacag ccgagtccca ggccctcctg gctaattcta
     3121 agtacaagaa agccaacaag agcttcctgc gggctaaaga gaatgccaca cacaacgtgc
     3181 gggtgtcccc taacacctct ctgtgcatta gactgctgaa ggacagcgcc ggaaaccaga
     3241 tgttcgacaa aatcggcaac gtgctcttcg gcatgcagat caaccacaag atcaccgtgg
     3301 gaaaacctaa ctacaagatc gaggtgggcg acagattcct gggcttcgat cagaaccaga
     3361 gcgagaacca cacctacgcc gtgctgcaga gagtgtccga gagcagtcac gacacccacc
     3421 actttaacgg ctgggacgtg aaggtgctgg aaaagggcaa agtgaccagc gatgtgatcg
     3481 tgcgggacga ggtctacgac caactgtctt acgagggcgt cccctacgat agcagcaagt
     3541 tcgccgagtg gcgggacaag cgcagaagat ttgtgcttga gaacctgagc atccagctgg
     3601 aagagggcaa gaccttcctg acagagttcg acaagctgaa taaggacagc ctgtaccgct
     3661 ggaacatgaa ctacctgaaa ctgctgagaa aggccatccg ggccggaggc aaagagttcg
     3721 ccaagatcgc taagacagag atcttcgagc tggcggtgga aagattcggc cctattaacc
     3781 tgggcagcct gtcccagatc agccttaaga tgattgcctc ctttaagggc gtggtccagt
     3841 cctacttctc cgtgagcggc tgcgtggatg atgcctccaa aaaggcccat gattctatgc
     3901 tgttcacatt tatgtgcgcc gccgaagaaa agcggaccaa caagagagaa gaaaagacca
     3961 acagagccgc cagctttatc ctgcaaaaag cctacctgca tggctgcaag atgatcgtgt
     4021 gcgaggacga ccttcctgtg gccgacggca agacaggcaa agcccagaat gccagacgga
     4081 tggactggtg cgccagagcc ctggccaaga aggtgaacga cggctgtgtt gccatgagca
     4141 tctgctacag agctatccct gcctacatga gcagccacca ggaccccttt gtgcacatgc
     4201 aggataagaa aaccagcgtg ctgcggccta gattcatgga agttaataag gatagcatca
     4261 gagactacca cgtggcgggc ctgagaagaa tgctgaacag caagagtgac gctggcacca
     4321 gtgtttatta ccggcaagct gccctgcatt tctgcgaagc cctgggcgtg agccctgaac
     4381 tggtgaaaaa caagaaaacc cacgccgccg aactgggcaa gcacatgggc agcgctatgc
     4441 tgatgccctg gagaggcggt agagtgtaca tcgccagcaa aaagctgacc tccgatgcca
     4501 aatcagtgaa gtactgcggc gaggatatgt ggcagtacca cgccgatgag atcgccgctg
     4561 ttaacatcgc catgtatgag gtgtgctgcc agaccggcgc tttcggaaag aaacagaaaa
     4621 aatcggacga gctgcctgga aaaaggccgg cggccacgaa gaaggccggc caggcaaaga
     4681 agaagaagta agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     4741 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4801 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4861 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     4921 tgctggggag cggccgctag ttattaatag taatcaatta cggggtcatt agttcatagc
     4981 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     5041 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     5101 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     5161 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     5221 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     5281 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     5341 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     5401 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     5461 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
     5521 cagatccgct agcatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     5581 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     5641 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     5701 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     5761 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     5821 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     5881 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     5941 cgacggcccc gtaatgcaga agaaaaccat gggctgggag gcctcctccg agcggatgta
     6001 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     6061 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     6121 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     6181 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     6241 gtaaggatcc gggatgcaga aattgatgat ctattaaaca ataaagatgt ccactaaaat
     6301 ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct acattttgaa
     6361 tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg cctgctcttt
     6421 actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat cataatttaa
     6481 acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta tagatctata
     6541 gatctctcgt gggatcattg tttttctctt gattcccact ttgtggttct aagtactgtg
     6601 gtttccaaat gtgtcagttt catagcctga agaacgagat cagcagcctc tgttccacat
     6661 acacttcatt ctcagtattg ttttgccaag ttctaattcc atcagaagct ggtcgacctg
     6721 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     6781 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     6841 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     6901 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     6961 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     7021 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     7081 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     7141 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     7201 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     7261 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     7321 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     7381 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     7441 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     7501 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     7561 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     7621 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     7681 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     7741 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     7801 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     7861 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     7921 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     7981 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     8041 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     8101 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     8161 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     8221 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     8281 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     8341 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     8401 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     8461 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     8521 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     8581 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     8641 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     8701 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     8761 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     8821 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     8881 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     8941 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     9001 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     9061 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     9121 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     9181 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     9241 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     9301 gctggccttt tgctcacatg t
//
