LOCUS       sequence-405070-        4964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSGKP-based plasmid containing a J23119-driven sgRNA cassette, with 
            replaceable spacer (BsaI-restriction sites). Kanamycin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4964)
  AUTHORS   Chen W, Ma J, Wu Z, Wang Z, Zhang H, Fu W, Pan D, Shi J, Ji Q
  TITLE     Cas12n nucleases, early evolutionary intermediates of type V CRISPR,
            comprise a distinct family of miniature genome editors.
  JOURNAL   Mol Cell. 2023 Jun 27:S1097-2765(23)00463-X. doi: 
            10.1016/j.molcel.2023.06.014.
  PUBMED    37402371
REFERENCE   2  (bases 1 to 4964)
  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: "Mol Cell. 
            2023 Jun 27:S1097-2765(23)00463-X. doi: 
            10.1016/j.molcel.2023.06.014."
COMMENT     Sequence Label: pSGKP_AcsgRNA
FEATURES             Location/Qualifiers
     source          1..4964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     432..448
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     433..449
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(511..1845,1846..1932))
                     /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        2318..2352
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     ncRNA           2573..2640
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(2713..2729)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2713..2729)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2788..2804)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2788..2804)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2801..2823)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2812..2828
                     /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(2836..2842,2843..2860,2861..2866))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2881..2902
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3019..3036)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3190..3778)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3270..3289)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3949..4764)
                     /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(3949..4764)
                     /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     4672..4691
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4765..4869)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4937..4955
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatggtac
      421 cgggcccccc ctcgaggtcg acggtatcga tacggcatca gagcagattg tactgagagt
      481 gcaccataat cggcattttc ttttgcgttt ttatttgtta actgttaatt gtccttgttc
      541 aaggatgctg tctttgacaa cagatgtttt cttgcctttg atgttcagca ggaagctagg
      601 cgcaaacgtt gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac
      661 gacattgttt cctttcgctt gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt
      721 aggatcaaga tccattttta acacaaggcc agttttgttc agcggcttgt atgggccagt
      781 taaagaatta gaaacataac caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt
      841 catttttgat ccgcgggagt cagtgaacag ataccatttg ccgttcattt taaagacgtt
      901 cgcgcgttca atttcatctg ttactgtgtt agatgcaatc agcggtttca tcactttttt
      961 cagtgtgtaa tcatcgttta gctcaatcat accgagagcg ccgtttgcta actcagccgt
     1021 gcgtttttta tcgctttgca gaagtttttg actttcttga cggaagaatg atgtgctttt
     1081 gccatagtat gctttgttaa ataaagattc ttcgccttgg tagccatctt cagttccagt
     1141 gtttgcttca aatactaagt atttgtggcc tttatcttct acgtagtgag gatctctcag
     1201 cgtatggttg tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta cattttgata
     1261 cgtttttccg tcaccgtcaa agattgattt ataatcctct acaccgttga tgttcaaaga
     1321 gctgtctgat gctgatacgt taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt
     1381 accggagaaa tcagtgtaga ataaacggat ttttccgtca gatgtaaatg tggctgaacc
     1441 tgaccattct tgtgtttggt cttttaggat agaatcattt gcatcgaatt tgtcgctgtc
     1501 tttaaagacg cggccagcgt ttttccagct gtcaatagaa gtttcgccga ctttttgata
     1561 gaacatgtaa atcgatgtgt catccgcatt tttaggatct ccggctaatg caaagacgat
     1621 gtggtagccg tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca
     1681 aacgtccagg ccttttgcag aagagatatt tttaattgtg gacgaatcga actcaggaac
     1741 ttgatatttt tcattttttt gctgttcagg gatttgcagc atatcatggc gtgtaatatg
     1801 ggaaatgccg tatgtttcct tatatggctt ttggttcgtt tctttcgcaa acgcttgagt
     1861 tgcgcctcct gccagcagtg cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt
     1921 tttgatgttc atcgttcatg tctccttttt tatgtactgt gttagcggtc tgcttcttcc
     1981 agccctcctg tttgaagatg gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg
     2041 taaggggtga cgccaaagta tacactttgc cctttacaca ttttaggtct tgcctgcttt
     2101 atcagtaaca aacccgcgcg atttactttt cgacctcatt ctattagact ctcgtttgga
     2161 ttgcaactgg tctattttcc tcttttgttt gatagaaaat cataaaagga tttgcagact
     2221 acgggcctaa agaactaaaa aatctatctg tttcttttca ttctctgtat tttttatagt
     2281 ttctgttgca tgggcataaa gttgcaagct tgatatcttg acagctagct cagtcctagg
     2341 tataatacta gtcagtgctg atcgatcgaa acgtcgcctg cgataggcgg gagacgctaa
     2401 acgcccgtgg agcatccata agaccaacca cctctcgggg cggtaggcac gacgcatcga
     2461 agcgggaagg ctccggcgct cggcctgagt cacctcagca gagtgatctg ctgacgctcc
     2521 caaccttgaa tgaaaacgcg tgtagggagc gatcgcgaga ccattggtct caggccggca
     2581 tggtcccagc ctcctcgctg gcgccggctg ggcaacatgc ttcggcatgg cgaatgggac
     2641 ccaattattg aacaccctaa cgggtgtttt tttgtttctg gtctaccgat atcgaattcc
     2701 tgcagcccgg gggatccact agttctagag cggccgccac cgcggtggag ctcatcccaa
     2761 tggcgcgccg agcttggctc gagaatggtc atagctgttt cctgtgtgaa attgttatcc
     2821 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2881 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2941 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3001 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3061 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3121 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3181 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3241 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3301 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3361 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3421 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3481 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3541 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3601 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3661 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3721 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3781 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3841 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3901 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt agaaaaactc
     3961 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcgatac catatttttg
     4021 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     4081 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     4141 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     4201 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     4261 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     4321 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     4381 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     4441 ctggaatgct gttttcccag ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     4501 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     4561 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     4621 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     4681 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     4741 cgtttcccgt tgaatatggc tcatactctt cctttttcaa tattattgaa gcatttatca
     4801 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4861 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     4921 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
