LOCUS       sequence-442958-        4727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of BaFnuc guide RNA .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4727)
  AUTHORS   Jiang K, Lim J, Sgrizzi S, Trinh M, Kayabolen A, Yutin N, Bao W, 
            Kato K, Koonin EV, Gootenberg JS, Abudayyeh OO
  TITLE     Programmable RNA-guided DNA endonucleases are widespread in 
            eukaryotes and their viruses.
  JOURNAL   Sci Adv. 2023 Sep 29;9(39):eadk0171. doi: 10.1126/sciadv.adk0171. 
            Epub 2023 Sep 27.
  PUBMED    37756409
REFERENCE   2  (bases 1 to 4727)
  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: "Sci Adv."; 
            date: "2023-09-29"; volume: "9(39):eadk0171. doi"; pages: " 
            10.1126/sciadv.adk0171. Epub 2023 Sep 27"
COMMENT     Sequence Label: pKJ223
FEATURES             Location/Qualifiers
     source          1..4727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     568..587
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        568..586
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    586..610
                     /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)."
     ncRNA           940..1007
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      1016..1063
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1388..1407)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1399..2214
                     /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             1399..2214
                     /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     complement(1472..1491)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2331..2348
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3710..3767)
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     rep_origin      join(4258..4727,1..119)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
       61 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      121 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      181 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
      241 ataccgctcg ccgcagccga acgaccgagc gcagcgaggt cggcgatata ggcgcgagca
      301 accgcccctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctgctca
      361 tgtttgacag cttatcatcg atgcataatg tgcctgtcaa atggacgaag cagggattct
      421 gcaaacccta tgctactccc tcgagccgtc aattgtctga ttcgttacca attatactag
      481 ctccggcaaa aaaacgggca aggtgtcacc accctgccct ttttctttaa aaccgaaaag
      541 attacttcgc gtttgccacc tgacgtctaa tacgactcac tatagggaat tgtgagcgga
      601 taacaattcc ccggatcccg ctcgagtgga gggaactgac aactgtagag tggagatcac
      661 cgacgaacgc tggacctcaa agacgtgtgg catgtgcaga tccatccacc gcgaacttgg
      721 ggcaaaagaa ctattcgagt gccccaactg ccactacacc tgccacagag acgtgcacgg
      781 agctcgaaac atcttgctga gaagcttcgg acagtttcca gtctagaaaa acacgaactt
      841 tttccttggc ccaaggattg ccaaacacca ctcaaatcct cgttcagggg cctcgagtgg
      901 ctgggcatat atgggttagc agccacctcc ttgttattgg gccggcatgg tcccagcctc
      961 ctcgctggcg ccggctgggc aacatgcttc ggcatggcga atgggacgcg gccgcctagc
     1021 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttggctagcg ctgaggtctg
     1081 cctcgtgaag aaggtgttgc tgactcatac caggcctgaa tcgccccatc atccagccag
     1141 aaagtgaggg agccacggtt gatgagagct ttgttgtagg tggaccagtt ggtgattttg
     1201 aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat ctgatccttc
     1261 aactcagcaa aagttcgatt tattcaacaa agccacgttg tgtctcaaaa tctctgatgt
     1321 tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac
     1381 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc caggccgcga
     1441 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     1501 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
     1561 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     1621 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     1681 tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct
     1741 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     1801 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     1861 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     1921 gttgaacaag tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc
     1981 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     2041 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     2101 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     2161 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa
     2221 ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga ctcgagatta
     2281 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     2341 tgagcgagga agcctgcata acgctgagag atcccctcat aatttcccca aagcgtaacc
     2401 atgtgtgaat aaattttgag ctagtagggt tgcagccacg agtaagtctt cccttgttat
     2461 tgtgtagcca gaatgccgca aaacttccat gcctaagcga actgttgaga gtacgtttcg
     2521 atttctgact gtgttagcct ggaagtgctt gtcccaacct tgtttctgag catgaacgcc
     2581 cgcaagccaa catgttagtt gaagcatcag ggcgattagc agcatgatat caaaacgctc
     2641 tgagctgctc gttcggctat ggcgtaggcc tagtccgtag gcaggacttt tcaagtctcg
     2701 gaaggtttct tcaatctgca ttcgcttcga atagatatta acaagttgtt tgggtgttcg
     2761 aatttcaaca ggtaagttag ttgctagaat ccatggctcc tttgccgacg ctgagtagat
     2821 tttaggtgac gggtggtgac aatgagtccg tgtcgagcgc tgattttttc ggcctttaga
     2881 gcgagattta tacaatagaa tttggcatga gattggattg cttttagtca gcctcttata
     2941 gcctaaagtc tttgagtgac tagatgacat atcatgtaag ttgctgatag gtttccagtt
     3001 ttccgctcct aggtctgcat attgtacttt tcctcttact cgacttaacc agtaccaacc
     3061 cagcttctca acggatttat accatggcac tttaaagcca gcatcactga caatgagcgg
     3121 tgtggtgtta ctcggtagaa tgctcgcaag gtcggctaga aattggtcat gagctttctt
     3181 tgaacattgc tctgaaagcg ggaacgcttt ctcataaaga gtaacagaac gaccgtgtag
     3241 tgcgactgaa gctcgcaata ccataagccg tttttgctca cggatatcag accagtcaac
     3301 aagtacaatg ggcatcgtat tgcccgaaca gataaagcta gcatgccaac ggtatacagc
     3361 gagtcgctct ttgtggaggt gacgattacc taacaatcgg tcgattcgtt tgatgttatg
     3421 ttttgttctc gctttggttg gcaggttacg gccaagttcg gtaagagtga gagttttaca
     3481 gtcaagtaag gcgtggcaag ccaacgttaa gctgttgagt cgttttaagt gtaattcggg
     3541 gcagaattgg taaagagagt cgtgtaaaat atcgagttcg cacattttgt tgtctgatta
     3601 ttgatttttg gcgaaaccat ttgatcatat gacaagatgt gtatctacct taacttaatg
     3661 attttgataa aaatcattag gggattcatc agtgcataac gcgaagtaat cttttcggtt
     3721 ttaaagaaaa agggcagggt ggtgacacct tgcccttttt tgccggactg cagcaatggc
     3781 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3841 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3901 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3961 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4021 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4081 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4141 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4201 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4261 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4321 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4381 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4441 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4501 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4561 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4621 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4681 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagc
//
