LOCUS       sequence-354819-        8304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3-component ShCAST containing a Cas12k-TniQ-TniQ fusion. 
            New sgRNA spacer sequences can be added using Golden Gate assembly 
            (via SapI sites)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8304)
  AUTHORS   Tou CJ, Orr B, Kleinstiver BP
  TITLE     Precise cut-and-paste DNA insertion using engineered type V-K 
            CRISPR-associated transposases.
  JOURNAL   Nat Biotechnol. 2023 Jan 2. doi: 10.1038/s41587-022-01574-x.
  PUBMED    36593413
REFERENCE   2  (bases 1 to 8304)
  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 
            Biotechnol. 2023 Jan 2. doi: 10.1038/s41587-022-01574-x."
COMMENT     Sequence Label: pShCAST(Cas12k-TniQ-TniQ)-sgRNA_entry (CJT12)
FEATURES             Location/Qualifiers
     source          1..8304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             5928..5939
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(join(5940..5946,5947..5964,5965..5970))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     rep_origin      complement(6070..6658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6150..6169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6829..7620,7621..7689))
                     /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     7452..7471
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7690..7794)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(7821..7848)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        complement(8103..8137)
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      complement(8151..8222)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tcccacagcg caggcatttt aaaaggtgct tgacagtttg gacactttgc taaaatcttg
       61 agttgatggc gatcgcactt ccacaccgac ttgtactgcc attcaattcg gtgacaaggc
      121 gactcggcat aacaagcccc acacaagcga attggctcat gctgcattcc cactccagca
      181 ggcggtaaca tctgggctaa cctttgagca tccacttcta ctacagatgc gatcgcttcc
      241 aattcttgct gactagggcg aggattaaaa tgaaatcttt cccaacgtgc cactatagca
      301 ccaattcctg ccaaagtacc caatccactt gcggataaat ggttggcacg tctgaacctg
      361 ccaagaaagt ggctcaggct ttccccttca tagggtttaa tcaagaatag ccaaggttta
      421 acatctggtg cttctataga gccaccgccg gacccacctc cagagccacc gccaaccttc
      481 tgtagctttg ccatttctgc aaacggcatc ctacatctgt gacagcaccc atcctcccac
      541 agcgcaggca ttttaaaagg tgcttgacag tttggacact ttgctaaaat cttgagttga
      601 tggcgatcgc acttccacac cgacttgtac tgccattcaa ttcggtgaca aggcgactcg
      661 gcataacaag ccccacacaa gcgaattggc tcatgctgca ttcccactcc agcaggcggt
      721 aacatctggg ctaacctttg agcatccact tctactacag atgcgatcgc ttccaattct
      781 tgctgactag ggcgaggatt aaaatgaaat ctttcccaac gtgccactat agcaccaatt
      841 cctgccaaag tacccaatcc acttgcggat aaatggttgg cacgtctgaa cctgccaaga
      901 aagtggctca ggctttcccc ttcatagggt ttaatcaaga atagccaagg tttaacatct
      961 ggtgcttcta tgctcccact ttcgggtgtg gcggactctg aggtcccggg agtctcgctg
     1021 ccgctacttc gcctagttag gcggagattg taagcagaaa gtgctaattc ctttgctttg
     1081 tcgtggggac tacctcgaat aggttgtttt ccctcctcaa tcacaattcc tgtttgagct
     1141 gctttacttt gaatgctttg aattaatctg ccgtagctcc accgatgaac attaacccgg
     1201 tactgtttgg catatttttg ctgaccttca atataaccgg gaaatttttg ttctgctata
     1261 gcttgaattt cactttggac aacctcccgc atatccccta acttgggcaa gacaatactg
     1321 ccagctttgt aggttctcgc taacgctaca attgctttag ctaataatct gtctatatgt
     1381 tgccctaact cagatgctcc aaattgattg ggagagaagt ttttttgtgc tttgtggcgt
     1441 tcgtgagata ggtactgctg ttgtcgtctc tggcgattta gcagttcgta attgtcgcca
     1501 agtaattgtt taatactccg gtaagccaag actttgttgg cgatcgcatc tactactgct
     1561 actgtggcag gtttttctag tcccaggctt actccaacca aaatgtgtga ttgaccttga
     1621 taaaggggtt ggctaggacg ctcaaaggaa ttgtttattc gagtaagtgt tgattgttta
     1681 cgttgaatca aagcttgctg tgtatcgctt agatcgcttt tcttcttcat gtttgtgatg
     1741 aatttagtaa tttcatctgc tttctcttgg cgaacgattt ctgttccctc ctctgtccac
     1801 aagcgattgt caacacagca gtaaagggtc aagtggtgaa gattccaagg ttccccttta
     1861 ccctcacctt caagccaaac tagatgacca tttctgagtg tgaacaagcc gctagaatgc
     1921 tgatttttgc ttttgcgttt agtctgttgg tcttctagga agcgttgaaa ccagtgaagt
     1981 tgacgattgc cgcagtacac ctcaaaaatt aaatcgctta agccattgaa gtgaacacac
     2041 agcctaccct tttgattctt tgaccaaacc atatcctcgt tggtttcaaa aacaaggggg
     2101 aatgggagag aacttgatcg agttaacaga atatcctgcc agcgtttggc ttgggcgttg
     2161 tcttcagcaa cagtggttgt agcagtcaag agtgtctcca accatttagc attggtcaaa
     2221 tctcgacctt taggcatccg acttattaac ttttcggtaa gcctttgaat ttggatttca
     2281 acttgacgac gacgtttagc aaatttttct gaatcttctt ctttgtcagt aagtttgcag
     2341 ccatttttta acaggtagct gatggcgcta cggctcttga tatcttccgt ttcttggtaa
     2401 gcgtcaaata atgtcttgga taaactacgc ttagggctag aagatgaggg ttttttctcc
     2461 tttttacctt ttttcccttt aggtgaagcg ctatctgact cagatgctgg catagctatt
     2521 gccaaaattt cagcagcttt gacacgaata gcctctaaag tgtcaccact aagttctact
     2581 aattcagcat cgctattgag catttctagc cagcgcgtct ttccatctag ctgctgttga
     2641 agccgtttct gtatagccag ccaggacttg tagatgtagt ccacaatatg aattgccgac
     2701 atatataaac ggctgggctg acctgcaaag cgagggtcag ttttgagagg ttgacataac
     2761 tggctcacaa cggtagacgg gagtttaccc ttttgttgcc acttctcgaa gtcggggtgt
     2821 tgacctaact ggcaaagcag ttcgttaatt aacggcgtgt ttaaatctgc catcaacttc
     2881 cagagttgtt ggcggtttga ttcaaaggaa ataagtcgag cttgaatagt tatttgactc
     2941 atgaaaaaat aatatcacat gaatacataa tgtgctttta aaaaatgtaa atttaaaaaa
     3001 ctacttctgt aggatgattt ctatggaagg aaaattctac acaagcacag aagcggctca
     3061 aatcactaat tgttcgcgtc gtcagctaca gtattggcgc gataaaggtg ttgtcgtgcc
     3121 aacagtgaat accacaggta aaggtcgtaa tgtttattac tcatatctga tctattggta
     3181 ttaaccggga tcccattaga gcaagttatc cgactcacgc cacatcaata gtaggaataa
     3241 tcttgccatg cgatcgcaca aattttttgg acaaccagat caattcaggg acttagttaa
     3301 tttcacacct ttttctggtt ttatcactgc agtcatttgt actccttagc tacttcctgt
     3361 aaaacagcct tgtcaatctt cttcaatcct cttgataagg aacgaattgc agcttcccta
     3421 agaatctcat caaggcgacc aatgtagcct tcagttgctg acgtgagaat ccgtagcatc
     3481 tccttgctct ttagattaga agatactggc agtttcaaaa ccatttgttc ccacatttct
     3541 acggtgttct taaaatcctc tcccgacaat ttaccaaagc gaagatgcgc ccgaaagcgt
     3601 tcgagaacct gctcatcccg cttaattacc gcatccaaac ggtctgttcc taccagtacc
     3661 acagcaattc ctaaatcttc ggcaatatct cgcacatcag caaaagtttc aggcttgaga
     3721 cggtcagctt catcaataat tagcatctct acgccacaac ccttgagtac ttctatcgtc
     3781 ctatctcgaa aatcagatac agtccctttt gttacccgat acttgaggta ctcagtaatc
     3841 tttttaaaca aatccttggg gccacatttt tggtgaggtc gaatataaac gacaggcaca
     3901 gttggaggtc gtccagcttc ctgctgaggt ttgtgcctgt atctgtaggc atcacaagca
     3961 actgtcttgc cagttctcga ttccccaact actcggcaag attttcttgc cttgcgcttg
     4021 ccatctaacc aatcatggag agtttttacc tgctgtaaag gcacaatgct cttacccttg
     4081 agacgagcaa tttcagcttg taaccactca tcatccggtt ttaccccacc caactgcttg
     4141 gcgatcgcct gagcttctgt cattgtttaa aacccatatt cttcacgaag ttgttcatag
     4201 tcccacacct caatgtcttc gtattgagag tgaaccgttt ctgtagattc cacttcatct
     4261 ggttcaacta tttgagaagg caaggattct ctattacttt catcaacagc agcagatcgc
     4321 aaaacagtct gttccaattt ttgacgctcc ttacggcttt tcttggtagc gacaagagca
     4381 tcgcggtcaa caacttcttg caataatgat tggttactga tagttttccc tgcggtacgg
     4441 agtctgcgac ttgctgcctc agcctcatct aatgccagtt gctctgtctc caaaccttga
     4501 gcgtgagcgc gagtcagaaa tacttcctga ttgttttcct ggcgataaac caaaattgtt
     4561 gtaatgtctc tggggtcaaa ccttaagttg acagtttctc cggcataacc tgccaaatat
     4621 tccccccgat acattaaatt ctgaaactgc aaacaaccac ctctttgcac agtgcgccgt
     4681 gactgcttca ttaaacaaat atccaaatct cgttctggta ttggtactgg cactgtaggc
     4741 aatcctgctt cccaacgctc aaagcgcgtt tggtcgccca tccgcgcatc aatactttgg
     4801 ttgtaacgat ctacgatgta acgcacaagt aactgttcta gttctcgcaa agtaagtctt
     4861 gcgtccttct ctgcatcttc tgggcgttcc tgcacattag atccggtgta cccaggaagc
     4921 gttgaaaata gttggtcatt taatgttttg aagggacgtt ctactactcc accttcagaa
     4981 gggcgatcgc gtaaatgaca gacaaatccc aattgcgccc caatctgact caagtggtta
     5041 gagcgaaagt ctttaccgcc atcagtataa aaatgttctg gttttccata ggttccccac
     5101 tcacaatgca gtttgtactc ggaaccgtaa cgctttggta gaattgcatg gcgtaacgct
     5161 aatgctacta ccccagaact gggtgcatca aagcccaagt tgatacccat aatgcaacga
     5221 gagtaagtat caattactgt tgttagccag ggacgactta aaatttcacc atgttgatct
     5281 accagcaaca catccacgcg ggtatggtca cattgccaaa catggttact gtaatcaacc
     5341 gataaatctt ttccttcacg ggttttaacc gaaagcgtag ttcctctcca accaggactg
     5401 cggatactct tggctttttg ttgcttttcc aaaatgggtg ctaatacccg taacacggtt
     5461 ttgtaattgg gcggcttaga gtcttttaat tcacgggctt tagcctcgac tctgagagca
     5521 acttgtttag gggtcatacg tttacttccc ttgttaccct ccttgtaggt tttggtaatg
     5581 aagttttccc aaaactcacc aatgcggtgt tttcctttat cagccctact tgtttgagtg
     5641 agtccgacta agccatcttg ttcccagttt ttcaccaacc tttgtaccgt tcgcaacgat
     5701 acatttagtt tctctgctgc ttcccgcaac ttttgcccat aagttgtgcg atcgcagggt
     5761 tccagtaaac tttggattac ctctagcttt acttgggctt cctctgagag ttgtgtggca
     5821 attacattct tctcaagagt tgtagcactt tctcctagta agccttccat aggaattgcc
     5881 aagggatgaa cagctaaatc aggattttgc tgactgttca tagatccttt ctcctctttc
     5941 aacatacgag ccggaagcat aaagtgtaaa attaataaga tgatcttctt gagatcgttt
     6001 tggagcttgg cgttgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6061 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6121 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6181 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6241 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6301 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6361 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6421 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6481 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6541 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6601 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6661 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6721 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6781 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6841 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6901 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6961 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     7021 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7081 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7141 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7201 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7261 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7321 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7381 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7441 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7501 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7561 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7621 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7681 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7741 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7801 tccccgaaaa gtgccacctg cgcagaaagg cccacccgaa ggtgagccga agagctcgag
     7861 atctcgctct tcctttcaac ccatttaggg ttcccccttg atcgggggtg attcagtagc
     7921 cactacagca gtacatccgc gcccttattg ctgggagcgc acctatcctg tctattgaca
     7981 gcagcatcag ggtgagcagc taccagggtc agaaagcaag actgtattta tacagtcaaa
     8041 ctaaccctca tttaacaaaa ttcagaggct gcaagcagca tgaattgcgg cgctattaat
     8101 atgctagcat tatacctagg actgagctag ctgtcagggt ctctctagta gagagcgttc
     8161 accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt
     8221 tgatgcctgg agatccttac tcaaaccttc tgtagctttg ccatttctgc aaacggcatc
     8281 ctacatctgt gacagcaccc atcc
//
