LOCUS       sequence-405896-        7013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CnCas12f1 with MS13 sgRNA site for Human cell genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7013)
  AUTHORS   Su M, Li F, Wang Y, Gao Y, Lan W, Shao Z, Zhu C, Tang N, Gan J, Wu 
            Z, Ji Q
  TITLE     Molecular basis and engineering of miniature Cas12f with C-rich PAM 
            specificity.
  JOURNAL   Nat Chem Biol. 2024 Feb;20(2):180-189. doi: 
            10.1038/s41589-023-01420-4. Epub 2023 Sep 11.
  PUBMED    37697004
REFERENCE   2  (bases 1 to 7013)
  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: 
            "10.1038/s41589-023-01420-4"; journalName: "Nat Chem Biol"; date: 
            "2024-02"; volume: "20"; issue: "2"; pages: "180-189"
COMMENT     Sequence Label: pCnCas12f1HS_sgRNA_MS13_empty
FEATURES             Location/Qualifiers
     source          1..7013
                     /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    513..630
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        679..1189
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1108..1126
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1211..1810
                     /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(1211..1230)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1707..1727
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1957..2078
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1968..1987)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2022..2041
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2326..2343)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2497..3085)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2577..2596)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3256..4047,4048..4116))
                     /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     3879..3898
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4117..4221)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4248..4264
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4248..4264
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4335..4559
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4548..4565
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4588..4605)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4614..4634)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6138..6158)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6174..6193)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6175..6193)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6235..6438)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6264..6284)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6439..6818
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    6915..7006
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6955..6974
                     /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 gacagggcga tttaacgtcc taaggctgag agaagtttat
      361 tctactcggc aagggttaat ctcgattgtt gtgttaccga tcgagcgttt cacaaatatg
      421 tgaaatgtaa atcgagaccc gagagagggt cggtctcctt ttattttttt tgaattctcg
      481 acctcgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
      541 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
      601 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
      661 ctttggccgc ggctcgaggg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc
      721 gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggac
      781 tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg
      841 ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc
      901 gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagtg
      961 ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc
     1021 agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt
     1081 agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac
     1141 gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagg gggatccacc
     1201 ggagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     1261 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     1321 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     1381 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     1441 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     1501 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     1561 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     1621 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     1681 cgccttcctg gaaacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     1741 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     1801 cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca
     1861 tgcatcggta cctttaagac caatgactta caaggcagct gtagatctta gccactttct
     1921 agagtcgggg cggccggccg cttcgagcag acatgataag atacattgat gagtttggac
     1981 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     2041 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     2101 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     2161 aatgtggtaa aatcgataag gatccgtcga ccgatgccct tgagagcctt caacccagtc
     2221 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     2281 atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc
     2341 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2401 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2461 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2521 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2581 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2641 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2701 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2761 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2821 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2881 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2941 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3001 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3061 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3121 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3181 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3241 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3301 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3361 cttaccatct ggccccagtg ctgcaatgat accgcgggac ccacgctcac cggctccaga
     3421 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3481 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3541 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3601 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3661 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3721 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3781 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3841 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3901 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3961 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4021 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4081 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4141 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4201 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgcag gaaacagcta
     4261 tgaccatgat tacgccaagc tctagctaga ggtcgacggt atcgagcccc agctggttct
     4321 ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca
     4381 atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag
     4441 acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag
     4501 ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt
     4561 cgaggctgat cagcgggttt aaactcaatg gtgatggtga tgatgaccgg ttagactttc
     4621 ctcttcttct tgggagaacc accagacacc atgctctcct tgattttgta atacttcgat
     4681 tcctccttct tggtgatgta cttgttgctc atggcgatgt ttctagcggc gttgtagtcg
     4741 gcgttcacct tgtagccgca cttcttgcag atgaagtcgg cttggctctc tctctgcccc
     4801 tcctcgtagt tgccgcactt gctgcaggtc tgagatgtgt ggtaggggtc gatgtacttc
     4861 accttgatgc cttctctctg agccttgtac tcgatcatgg tctgcagctg atagtagctc
     4921 cagtttctca ggatgctttt attctgtgtc tccttcaggc tcagcagctc catgttgatc
     4981 tgctcggctt ggttgctcac ggcgaacttc acgatgttgc tgctcaggaa gtggttgtag
     5041 gtcttggcga agttcttctc cttctcggcg aatctctcca gggcttgcag cttgttcact
     5101 ctgcccttgc cgcccttggc gctctgaatg gcgatctgca gtctccgtct gcggctctgc
     5161 atctgtgttc tcaccttcag gaagtcgtcg attctgccga tgctcttctt gatgtactcc
     5221 acgtcgttca gggcgcagta ggcggggatc ttcaggccca ggtccacgcc caccactctg
     5281 ccgctcacct tcttgttcac gatgtcgatg gggatgtcca gggtcaggtt caggatcagg
     5341 ttgttgtttt tatcaaactg caggctgctg tcgcagatct tgtacttgcc ctcgatcacc
     5401 ttgtgcaggg tgtgtctcag ctccaggctg ttcttgattc tgttgcccag gatcactttg
     5461 aatttgatgc cctccatcca cttgatctcg atgtcgtcgt tgtcgtcgta cttgaacttc
     5521 aggtcccggc ctcttgtcat cagggggaag tttctcttgt agtttctgat gcttctctcg
     5581 cccttcagca ggccgttgct cttgtcgctc ttgatgtcgg ccttgattct ctgtgtcacc
     5641 aggctcttgt tgtccaggtg cttgaagggg atcttgtcca gcacttggta caggtcgtcc
     5701 acgtacttct ccttgaactc cttctgaatg tccattcttt ctttctgtgt ttcttccagg
     5761 atcttgttca gctccacgat cttcagcttg tagtcctcga tcttgttctc caccaggctg
     5821 atcttatact tgatctcgtc cagcttctcg ctgctgctgt tcttcttgtt ctgctccttc
     5881 tccagtctag cggtcagctt ctcgatctcc ttcttatttt tgtctatgtc cttctcgcag
     5941 tccttcaggt ccttgatggt tctgatctcc aggccgcttt cgttcatgta caggatgtcg
     6001 ttcacggcca ggtaggtcat gcccatgttc agggctctgt actgattgta ctgctcgtcc
     6061 cggatccact tgtactgaga gtttcttgtg tccttgtcgc ccatgatggt cagcttgatc
     6121 tttctcacgg tgatcatgac tttcctcttc ttcttgggca tggtggcggc tctccctata
     6181 gtgagtcgta ttagcggccg cggatctcta gcggatctga cggttcacta aaccagctct
     6241 gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt
     6301 gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc
     6361 aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta
     6421 ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt
     6481 aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt
     6541 gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt
     6601 ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg
     6661 aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc
     6721 atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt
     6781 aattgattac tattaataac tagtcaataa tcaatgtcaa cgcgtatatc tggcccgtac
     6841 atcgcgaagc agcgcaaaac gcctaaccct aagcagattc ttcatgcaat tgtcggtcaa
     6901 gaatcgatag tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca
     6961 aaataggctg tccccagtgc aagtgcaggt gccagaacat ttctctatcg ata
//
