LOCUS       sequence-470084-        7338 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for sgRNA-S1b and eCas12f1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7338)
  AUTHORS   Park SJ, Ju S, Jung WJ, Jeong TY, Yoon DE, Lee JH, Yang J, Lee H, 
            Choi J, Kim HS, Kim K
  TITLE     Robust genome editing activity and the applications of enhanced 
            miniature CRISPR-Cas12f1.
  JOURNAL   Nat Commun. 2025 Jan 15;16(1):677. doi: 10.1038/s41467-025-56048-w.
  PUBMED    39809780
REFERENCE   2  (bases 1 to 7338)
  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: "Nat 
            Commun."; date: "2025-01-15"; volume: "16(1):677. doi"; pages: " 
            10.1038/s41467-025-56048-w"
COMMENT     Sequence Label: pU6-sgRNA-S1b (BsaI)-CBh-eCas12f1
FEATURES             Location/Qualifiers
     source          1..7338
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(88..107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        738..842
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(843..911,912..1703)
                     /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(1061..1080)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1874..2462
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2363..2382
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2524..2772
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2524..2544
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2695..2714
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(3120..3443)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     3338..3360
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3352..3369
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3353..3369
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3370..3426
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3439..3455)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3439..3455)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3452..3474)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3463..3479
                     /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(3487..3493,3494..3511,3512..3517))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     enhancer        3572..3857
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          4137..4364
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             4418..4438
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6035..6082
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             6092..6148
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(6149..6151,6152..6154,6155..6868)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6149..6151,6152..6154,6155..6865)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6194..6215)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6455..6474)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6802..6823
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6902..6919)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6908..7115
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7124..7264
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7124..7253
                     /label=AAV2 ITR
ORIGIN
        1 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
      361 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
      481 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
      541 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
      601 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
      661 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
      721 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      781 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
      841 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
      901 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
      961 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     1021 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1081 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1141 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1201 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1261 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1321 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     1381 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     1441 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     1501 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     1561 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     1621 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     1681 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     1741 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     1801 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1861 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1921 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1981 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     2041 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2101 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2161 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2221 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2281 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2341 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2401 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2461 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2521 tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     2581 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     2641 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     2701 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     2761 ggacgaaaca ccgaccgctg gacttcgagt ccaggtgggc tgcttgcatc agcctaatgt
     2821 cgagaagtgc tttcttcgga aagtaaccct cgaaacaaag aaaggaatgc aacggagacc
     2881 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     2941 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     3001 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3061 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     3121 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     3181 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
     3241 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     3301 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     3361 acggccagtg aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggcatgc
     3421 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3481 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatagatccg
     3541 gtctcctttt attttttgtt ttagaggtac ccgttacata acttacggta aatggcccgc
     3601 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt
     3661 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     3721 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     3781 attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     3841 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
     3901 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
     3961 gggcgggggg gggggggggg cgcgcgccgg gggggggggg ggggggsggg ggggggggsg
     4021 gggsgrggsg grgrggkgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
     4081 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
     4141 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
     4201 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
     4261 gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa
     4321 tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac
     4381 catgaaacgg acagccgacg gaagcgagtt cgagtcacca aagaagaagc ggaaagtcgc
     4441 caagaacaca atcaccaaga cactgaagct gagaatcgtg cggccttaca acagcgccga
     4501 ggtggaaaaa atcgtggccg atgagaagaa caacagagag aaaatcgctc tggaaaaaaa
     4561 taaggacaag gttaaagaag cctgctccaa gcacctgaaa gtggctgcct actgcaccac
     4621 ccaggtcgag agaaacgcct gtctgttctg caaggccaga aagctggacg acaagttcta
     4681 ccaaaagctg agaggccagt tccccgacgc cgtgttctgg caggagatta gcgagatttt
     4741 cagacagctg cagaagcagg ccgccgagat ctacaaccag agcctgatcg agctgtacta
     4801 cgagatcttt atcaaaggca aaggaattgc caacgcttct agcgtggaac actacctgag
     4861 cagagtgtgc taccggagag ccgccgagct gttcaagaat gccgctatcg ccagcggact
     4921 gcggagcaag atcaagtcta acttccggct gaaggaactc aagaatatga aatcccggct
     4981 gccaacaaca aagagcgaca actttccaat ccccctggtg aagcagaaag gaggccagta
     5041 taccggcttt gagatcagca accacaactc tgatttcatc atcaagatcc cattcggcag
     5101 atggcaagtg aagaaggaaa tcgacaagta cagaccttgg gagaagttcg acttcgagca
     5161 ggtgcagaag agtcctaagc ctatcagcct gctgctgtct acccagagac ggaaacggaa
     5221 caaaggctgg tccaaggacg agggcaccga agccgaaatc aagaaggtga tgaacggcga
     5281 ctaccagacc agctacatcg aagtgaagag gggcagcaag atcggcgaga agtctgcctg
     5341 gatgctgaac ctgagcatcg atgttcctaa gatcgacaaa ggagtggacc ccagcatcat
     5401 cggcggaatt gatgtgggcg tgagaagccc tctggtctgt gccatcaaca atgcttttag
     5461 cagatacagc atctccgata acgacctgtt tcactttaac aagaagatgt tcgccaggcg
     5521 gagaatcctg cttaaaaaga accgccacaa gagagccggc cacggcgcca agaacaagct
     5581 caaacctatc accatcctga ccgagaagag cgagcggttc cggaaaaagc tgatcgagag
     5641 atgggcctgt gaaatcgccg acttcttcat caaaaataag gtgggcacag tgcagatgga
     5701 aaacctggaa ggcatgaaga gaaaggaaga tagctatttc aacatccggc tgagaggctt
     5761 ctggccttac gccgagatgc agaacaagat cgagttcaag ctgaagcaat acggcatcga
     5821 gatcagaaag gtggctccta acaacaccag caaaacctgt agcaagtgcg gccatctgaa
     5881 caactacttc aacttcgagt acagaaagaa gaacaagttc ccccacttca agtgcgagaa
     5941 gtgcaacttc aaggaaaacg ccgattataa tgctgcgctg aatatctcta atcctaagct
     6001 gaaatctaca aaggaggaac ccggaagcgg atccgattac aaagatgacg atgacaaaga
     6061 ctacaaggat gatgatgata agggatccgg cgcaacaaac ttctctctgc tgaaacaagc
     6121 cggagatgtc gaagagaatc ctggaccgat ggtgagcaag ggcgaggagc tgttcaccgg
     6181 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     6241 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     6301 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     6361 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     6421 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     6481 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     6541 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     6601 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     6661 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     6721 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     6781 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     6841 tctcggcatg gacgagctgt acaagtaaac gcgttaagaa ttcctagagc tcgctgatca
     6901 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     6961 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     7021 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     7081 gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg
     7141 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     7201 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     7261 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     7321 cgtcaaagca accatagt
//
