LOCUS       sequence-418047-        5403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express KnFnuc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5403)
  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 5403)
  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: pKJ237
FEATURES             Location/Qualifiers
     source          1..5403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1999..2017)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2013..2060
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2426..2431,2432..2448,2449..2454))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2475..2491)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2475..2491)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2488..2510)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2499..2515
                     /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(2523..2529,2530..2547,2548..2553))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2568..2589
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2706..2723)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2877..3465)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2957..2976)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3636..4427,4428..4496))
                     /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     4259..4278
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4497..4601)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4669..4687
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4725..4747)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4847..4866
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5062..5084
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5076..5093
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5077..5093
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5213..5232
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5316..5335
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5316..5334
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             5366..5388
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tcccaccatc atcatcatca ctctagtggc atggatgaag gggcggacga tagcgaagag
       61 gctaagagaa aaagacccga tatcactctg cggcgcgcgt tgcgcaaaga taaggaaacc
      121 agtgttgtcc aaacaggatg gaagttttta tgccaggaac ttggcatacg tgaccgtata
      181 gaagaaatta ttcctgaggt cacacggatc cgggtcgaga cgtgtttgct gttgaactta
      241 cacttcatcc gcttattaga cgaaggccgc ccgatccctg tgatagacca aaaccttgta
      301 gggagagcaa tgcaatgtac atacagtaag aacccgcagg cggatccaga cctgcacgag
      361 actttcgtac atcattattt acctttgtgc ccgaatcgcc ccaacaactc ctgcttaccg
      421 cgtattacaa acgtgctgct ggacctgcgc aaccaattgc ttagcaatat aaagaatcac
      481 gtagctgtcc tgttccagag tcgccatcgc gcttttatga agttgttact gcgtgaggcc
      541 gctccagatg tcccattttt cggagacgcg gatgaggatc ttgaatcttg cacacgtctg
      601 ttaacgactg ctacattgtg gcgccccaac gagtccgtac gtgagttact tccggaatat
      661 cccagaatct acggcagaat acccgaagcg gctatagagt gcctgcagga tttagttgac
      721 tctgttagac cggaggttgg tccgctgccc gcggccccgc aatctcgccc tcatctttac
      781 atgccgtgga tgcggattat aagcgaagag tttagcgacc gcgagttgag atcattcagc
      841 ttggtccccc atgcatcatt cagcgcgcca ttcattgcta tcacgccgac gacgtggcca
      901 gaactgcagc ctaagtcagg aaagagaaaa gcgccgggtg agttgcgcga tgcttttccg
      961 agcataggcc gcctggaatc tggtggaaag acgtttgctg atcggataac aactgacgga
     1021 gtatctgcat ctgtttactt cttagtagag aagcgtactc ctcctccaga ggaccgcgtc
     1081 gttcatattc atccaaaaca gagagtagtt gggcttgatc caggcaagca cccggatttt
     1141 ctgaccggca tagctgtcac aggagactgg gatgggattg aacgccagga agaaatcata
     1201 ggcctgggca cgagagattt ctatcaccgg gcgggcttta agaaacgtac ctttctgatg
     1261 cacagttgga tgtcgcgtga tctggatgta gcggcgttca acaaagacgc gccaagcgga
     1321 aacacagtct ctttagagga ctttggaaaa cgcgttacgt tcgtctgcgc caacttgtac
     1381 gtccttgtaa gatttcatac ggccagacgc gtccggaaat tacgtcggag agtcaccatt
     1441 aagaaacaaa ttgaggtgga cagagcatgt aagagaataa cagctggtaa aaagacagtg
     1501 gtagcattcg gtgcggcgca ggtgtgggca gggagaacta agcgtcaatg tgggccgtgc
     1561 gaatcggtga agagaagatt gtcctcacac cataaagcca cggtggtgat gatcgacgaa
     1621 tttcgcacct cgcaggtatg tagcacctgt catagtgacg tcggcaagtt cgcagtgttg
     1681 aagcgccagc gggtaatgga agacggtctt ccgacagtta ccgaaggggg acggcgtgag
     1741 gatgaggacg aagacggagg aggacggaca tcgtacaaaa cttgtcacaa tgtgagagcg
     1801 tgtactaacc ctttatgccg catggtgtgg aatcgtgacg tgaatgcagc tagaaatatc
     1861 gcatggattt gcatgagtat tgccagaggc gaaggtcggc ctgcggagtt caccagagca
     1921 ggtgtttggg gttaactcga gccttaggag atccggctgc taacaaagcc cgaaaggaag
     1981 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2041 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatcca caggacgggt
     2101 gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg
     2161 cggcggccaa agcggtcgga cagtgctccg agaacgggtg cgcatagaaa ttgcatcaac
     2221 gcatatagcg ctagcagcac gccatagtga ctggcgatgc tgtcggaatg gacgatatcc
     2281 cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg
     2341 gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag
     2401 caatttaact gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga
     2461 gaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     2521 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     2581 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2641 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2701 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2761 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2821 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2881 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2941 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3001 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3061 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3121 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3181 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3241 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3301 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3361 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3421 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3481 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3541 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3601 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3661 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3721 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3781 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3841 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3901 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3961 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4021 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4081 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4141 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4201 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4261 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4321 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4381 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4441 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4501 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4561 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4621 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     4681 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     4741 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     4801 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     4861 attgtactga gagtgcacca tatatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4921 aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     4981 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     5041 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgccaagc
     5101 ttgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     5161 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     5221 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     5281 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta tagggagacc
     5341 acaacggttt ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggg
     5401 tca
//
