LOCUS       sequence-404780-        6816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AsCas12f1-v5.1+T1-based Human cell genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6816)
  AUTHORS   Wu Z, Liu D, Pan D, Yu H, Shi J, Ma J, Fu W, Wang Z, Zheng Z, Qu Y, 
            Li F, Chen W, Huang X, Shen H, Ji Q
  TITLE     Structure and engineering of miniature Acidibacillus sulfuroxidans 
            Cas12f1
  JOURNAL   Nature Catalysis (2023) 6, 695–709
REFERENCE   2  (bases 1 to 6816)
  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: "Nature 
            Catalysis (2023) 6, 695–709"
COMMENT     Sequence Label: pCMV-AsCas12f1-v5.1+T1-empty
FEATURES             Location/Qualifiers
     source          1..6816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    538..655
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        704..1214
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1133..1151
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1236..1835
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1236..1255)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1732..1752
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1982..2103
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1993..2012)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2047..2066
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2351..2368)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2522..3110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2602..2621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3281..4072,4073..4141))
                     /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     3904..3923
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4142..4246)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4273..4289
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4273..4289
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4360..4584
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4573..4590
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4613..4630)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4639..4659)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5938..5958)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      5961..5970
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5977..5996)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5978..5996)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6038..6241)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6067..6087)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6242..6621
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    6718..6809
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6758..6777
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gattcgtcgg ttcagcgacg ataagccgag aagtgccaat
      361 aaaactgtta agtggtttgg taacgctcgg taaggtagcc aaaaggctga aactccgtgc
      421 acaaagaccg cacggacgct tcacatactt gtggagtgtg aaccgagacc cgagagaggg
      481 tcggtctcct ttttttgaat tctcgacctc gagacaaatg gcagtattca tccacaattt
      541 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
      601 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
      661 ttattacagg gacagcagag atccactttg gccgcggctc gagggggttg gggttgcgcc
      721 ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga
      781 aacgcagcgg cgccgaccct gggactcgca cattcttcac gtccgttcgc agcgtcaccc
      841 ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc
      901 gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca
      961 ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg
     1021 ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg
     1081 tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg
     1141 cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg
     1201 acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag cccacggtgc
     1261 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     1321 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     1381 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     1441 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     1501 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     1561 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     1621 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     1681 aggcggccga gcgcgccggg gtgcccgcct tcctggaaac ctccgcgccc cgcaacctcc
     1741 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     1801 gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc
     1861 cgaccgaaag gagcgcacga ccccatgcat cggtaccttt aagaccaatg acttacaagg
     1921 cagctgtaga tcttagccac tttctagagt cggggcggcc ggccgcttcg agcagacatg
     1981 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     2041 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     2101 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     2161 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc gtcgaccgat
     2221 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     2281 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     2341 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2401 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2461 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2521 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2581 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2641 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2701 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2761 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2821 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2881 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2941 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3001 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3061 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3121 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3181 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3241 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3301 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3361 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3421 gggacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3481 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3541 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3601 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3661 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3721 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3781 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3841 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3901 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3961 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4021 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4081 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4141 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4201 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4261 aagtgccacc tgcaggaaac agctatgacc atgattacgc caagctctag ctagaggtcg
     4321 acggtatcga gccccagctg gttctttccg cctcagaagc catagagccc accgcatccc
     4381 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
     4441 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
     4501 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
     4561 atggctggca actagaaggc acagtcgagg ctgatcagcg ggtttaaact caatggtgat
     4621 ggtgatgatg accggttaga ctttcctctt cttcttggga gaaccaccag acttaatgct
     4681 ctcggcgatg atcttgtcga tgttggggat ggcgatattc cgggcggcgt tgtagtcggc
     4741 gttggcctcg tagccgcagg cccggcactt aaagatggcc tggccgattc tgttgccgga
     4801 gtcgatgttg ccgcactcgg agcatctctg gctggtgtac tggggtctga tcttgatcac
     4861 tttgatgccg gcctcctcgg ccttgtagat gatcttctgc tgcaggtcgt agtaggtcca
     4921 gttctgcagg aatctgctgc cgatgtctct gatgtttgtc agatcctcca tctggattgt
     4981 gccgcagccc tccttgatgg ccatgtccac gatgtatctg ctgtaccggt gattggtggt
     5041 gtctctgaaa ttggcgatct tatccctcag ctgctcaatg ggcttgattc tcttgtctct
     5101 gccgtggccg cccctggcgc cgccggcgta cttgccctgt ctcagcatgg agatgcgccg
     5161 gctctccacc tgcctcctga agttctcaat ctcgccgccc tccagcttgt acctggcggg
     5221 ggtgtgctgg aaggccatgt acacggccac ggccacgccc aggtcaatgc ccatgatctt
     5281 gttcaggtcc agcactctgg tctgtggctc gaagtcgtag ctgatgttca ggtaccactt
     5341 gttcttccgg tcgtcgtgga taatctggct ggcgctcacc tggtactcgc cggacaggat
     5401 tctgtccatg atggtcttgc cggcgcccct cacgatgatg atcacggaga tctttctctt
     5461 cacgttcatc tcctgcttgg cggggttgct cagcagagac aggctggcga tgtagtcgcc
     5521 gtgattcatc ctgttcacgg agatgttctc cttgatcagg tccaggggga tgtccctctt
     5581 gtaggcgggg atagacatgt cgccgcgcag gatctccttc tggtaccttc tgaacctgtc
     5641 tgtggctctc ttgatggtct ggctcagatt tccagagttc agtctgtagg cctttgtctt
     5701 gatggtgtgg taggcgtagc cgtgcacctg ggtgtagccc aggatgtcct tgctcttggg
     5761 gtactcgccg tggttatcct tgtagtcgct gctgaagccc atccactccc aggccagctg
     5821 tgtggcctta ttcagggcga atctggtttc ctgctgcagc tgtctcagga tggtgccgaa
     5881 ctccttccag tccaggtcca gaggcttcac gatctcgtat ctgtacacct tgatcatgac
     5941 tttcctcttc ttcttggggg ccatggtggc ggctctccct atagtgagtc gtattagcgg
     6001 ccgcggatct ctagcggatc tgacggttca ctaaaccagc tctgcttata tagacctccc
     6061 accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa
     6121 agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg
     6181 gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca
     6241 ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg
     6301 tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg
     6361 tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca
     6421 cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg
     6481 acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg
     6541 taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat
     6601 aactagtcaa taatcaatgt caacgcgtat atctggcccg tacatcgcga agcagcgcaa
     6661 aacgcctaac cctaagcaga ttcttcatgc aattgtcggt caagaatcga tagtactaac
     6721 atacgctctc catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag
     6781 tgcaagtgca ggtgccagaa catttctcta tcgata
//
