LOCUS       sequence-341488-        4943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA_v1 plasmid for AsCas12f1 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4943)
  AUTHORS   Wu Z, Zhang Y, Yu H, Pan D, Wang Y, Wang Y, Li F, Liu C, Nan H, Chen
            W, Ji Q
  TITLE     Programmed genome editing by a miniature CRISPR-Cas12f nuclease.
  JOURNAL   Nat Chem Biol. 2021 Sep 2. pii: 10.1038/s41589-021-00868-6. doi: 
            10.1038/s41589-021-00868-6.
  PUBMED    34475565
REFERENCE   2  (bases 1 to 4943)
  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 Chem 
            Biol. 2021 Sep 2. pii: 10.1038/s41589-021-00868-6. doi: 
            10.1038/s41589-021-00868-6."
COMMENT     Sequence Label: psgRNAv1-empty
FEATURES             Location/Qualifiers
     source          1..4943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(942..1757)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(942..1757)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1665..1684
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1758..1862)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1930..1948
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1986..2008)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2321..2343
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2335..2352
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2336..2352
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2389..2405
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2390..2406
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(2468..3802,3803..3889))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        4275..4309
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     ncRNA           4498..4565
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      4576..4623
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4649..4665)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4649..4665)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4724..4740)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4724..4740)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4737..4759)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4748..4764
                     /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(4772..4778,4779..4796,4797..4802))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4817..4838
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttagaaaaa ctcatcgagc
      961 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1021 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1081 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     1141 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     1201 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     1261 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     1321 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     1381 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     1441 gctgttttcc cagggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     1501 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     1561 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     1621 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     1681 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     1741 cgttgaatat ggctcatact cttccttttt caatattatt gaagcattta tcagggttat
     1801 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     1861 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     1921 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     1981 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     2041 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     2101 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     2161 cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac
     2221 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     2281 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     2341 acgacggcca gtgaattgac gcgtattggg atggtaccgg gccccccctc gaggtcgacg
     2401 gtatcgatac ggcatcagag cagattgtac tgagagtgca ccataatcgg cattttcttt
     2461 tgcgttttta tttgttaact gttaattgtc cttgttcaag gatgctgtct ttgacaacag
     2521 atgttttctt gcctttgatg ttcagcagga agctaggcgc aaacgttgat tgtttgtctg
     2581 cgtagaatcc tctgtttgtc atatagcttg taatcacgac attgtttcct ttcgcttgag
     2641 gtacagcgaa gtgtgagtaa gtaaaggtta catcgttagg atcaagatcc atttttaaca
     2701 caaggccagt tttgttcagc ggcttgtatg ggccagttaa agaattagaa acataaccaa
     2761 gcatgtaaat atcgttagac gtaatgccgt caatcgtcat ttttgatccg cgggagtcag
     2821 tgaacagata ccatttgccg ttcattttaa agacgttcgc gcgttcaatt tcatctgtta
     2881 ctgtgttaga tgcaatcagc ggtttcatca cttttttcag tgtgtaatca tcgtttagct
     2941 caatcatacc gagagcgccg tttgctaact cagccgtgcg ttttttatcg ctttgcagaa
     3001 gtttttgact ttcttgacgg aagaatgatg tgcttttgcc atagtatgct ttgttaaata
     3061 aagattcttc gccttggtag ccatcttcag ttccagtgtt tgcttcaaat actaagtatt
     3121 tgtggccttt atcttctacg tagtgaggat ctctcagcgt atggttgtcg cctgagctgt
     3181 agttgccttc atcgatgaac tgctgtacat tttgatacgt ttttccgtca ccgtcaaaga
     3241 ttgatttata atcctctaca ccgttgatgt tcaaagagct gtctgatgct gatacgttaa
     3301 cttgtgcagt tgtcagtgtt tgtttgccgt aatgtttacc ggagaaatca gtgtagaata
     3361 aacggatttt tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt gtttggtctt
     3421 ttaggataga atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg ccagcgtttt
     3481 tccagctgtc aatagaagtt tcgccgactt tttgatagaa catgtaaatc gatgtgtcat
     3541 ccgcattttt aggatctccg gctaatgcaa agacgatgtg gtagccgtga tagtttgcga
     3601 cagtgccgtc agcgttttgt aatggccagc tgtcccaaac gtccaggcct tttgcagaag
     3661 agatattttt aattgtggac gaatcgaact caggaacttg atatttttca tttttttgct
     3721 gttcagggat ttgcagcata tcatggcgtg taatatggga aatgccgtat gtttccttat
     3781 atggcttttg gttcgtttct ttcgcaaacg cttgagttgc gcctcctgcc agcagtgcgg
     3841 tagtaaaggt taatactgtt gcttgttttg caaacttttt gatgttcatc gttcatgtct
     3901 ccttttttat gtactgtgtt agcggtctgc ttcttccagc cctcctgttt gaagatggca
     3961 agttagttac gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc caaagtatac
     4021 actttgccct ttacacattt taggtcttgc ctgctttatc agtaacaaac ccgcgcgatt
     4081 tacttttcga cctcattcta ttagactctc gtttggattg caactggtct attttcctct
     4141 tttgtttgat agaaaatcat aaaaggattt gcagactacg ggcctaaaga actaaaaaat
     4201 ctatctgttt cttttcattc tctgtatttt ttatagtttc tgttgcatgg gcataaagtt
     4261 gcaagcttga tatcttgaca gctagctcag tcctaggtat aatactagta ttcgtcggtt
     4321 cagcgacgat aagccgagaa gtgccaataa aactgttaag tggtttggta acgctcggta
     4381 aggtagccaa aaggctgaaa ctccgtgcac aaagaccgca cggacgcttc acatatagct
     4441 cataaacaag ggtttgcgag ctagcttgtg gagtgtgaac cgagaccatt ggtctcaggc
     4501 cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa catgcttcgg catggcgaat
     4561 gggacgatct gacagctagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     4621 ttggatatcg aattcctgca gcccggggga tccactagtt ctagagcggc cgccaccgcg
     4681 gtggagctca tcccaatggc gcgccgagct tggctcgagc atggtcatag ctgtttcctg
     4741 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4801 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4861 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4921 gaggcggttt gcgtattggg cgc
//
