LOCUS       sequence-354706-        7770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3-component ShCAST containing a Cas12k-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 7770)
  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 7770)
  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)-sgRNA_entry (CJT11)
FEATURES             Location/Qualifiers
     source          1..7770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(57..62,63..80,81..87)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             88..99
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5575..5646
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        5660..5694
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      5949..5976
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        6003..6107
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6108..6176,6177..6968)
                     /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     complement(6326..6345)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7139..7727
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7628..7647
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggccttttgc tcaacgccaa gctccaaaac gatctcaaga agatcatctt attaatttta
       61 cactttatgc ttccggctcg tatgttgaaa gaggagaaag gatctatgaa cagtcagcaa
      121 aatcctgatt tagctgttca tcccttggca attcctatgg aaggcttact aggagaaagt
      181 gctacaactc ttgagaagaa tgtaattgcc acacaactct cagaggaagc ccaagtaaag
      241 ctagaggtaa tccaaagttt actggaaccc tgcgatcgca caacttatgg gcaaaagttg
      301 cgggaagcag cagagaaact aaatgtatcg ttgcgaacgg tacaaaggtt ggtgaaaaac
      361 tgggaacaag atggcttagt cggactcact caaacaagta gggctgataa aggaaaacac
      421 cgcattggtg agttttggga aaacttcatt accaaaacct acaaggaggg taacaaggga
      481 agtaaacgta tgacccctaa acaagttgct ctcagagtcg aggctaaagc ccgtgaatta
      541 aaagactcta agccgcccaa ttacaaaacc gtgttacggg tattagcacc cattttggaa
      601 aagcaacaaa aagccaagag tatccgcagt cctggttgga gaggaactac gctttcggtt
      661 aaaacccgtg aaggaaaaga tttatcggtt gattacagta accatgtttg gcaatgtgac
      721 catacccgcg tggatgtgtt gctggtagat caacatggtg aaattttaag tcgtccctgg
      781 ctaacaacag taattgatac ttactctcgt tgcattatgg gtatcaactt gggctttgat
      841 gcacccagtt ctggggtagt agcattagcg ttacgccatg caattctacc aaagcgttac
      901 ggttccgagt acaaactgca ttgtgagtgg ggaacctatg gaaaaccaga acatttttat
      961 actgatggcg gtaaagactt tcgctctaac cacttgagtc agattggggc gcaattggga
     1021 tttgtctgtc atttacgcga tcgcccttct gaaggtggag tagtagaacg tcccttcaaa
     1081 acattaaatg accaactatt ttcaacgctt cctgggtaca ccggatctaa tgtgcaggaa
     1141 cgcccagaag atgcagagaa ggacgcaaga cttactttgc gagaactaga acagttactt
     1201 gtgcgttaca tcgtagatcg ttacaaccaa agtattgatg cgcggatggg cgaccaaacg
     1261 cgctttgagc gttgggaagc aggattgcct acagtgccag taccaatacc agaacgagat
     1321 ttggatattt gtttaatgaa gcagtcacgg cgcactgtgc aaagaggtgg ttgtttgcag
     1381 tttcagaatt taatgtatcg gggggaatat ttggcaggtt atgccggaga aactgtcaac
     1441 ttaaggtttg accccagaga cattacaaca attttggttt atcgccagga aaacaatcag
     1501 gaagtatttc tgactcgcgc tcacgctcaa ggtttggaga cagagcaact ggcattagat
     1561 gaggctgagg cagcaagtcg cagactccgt accgcaggga aaactatcag taaccaatca
     1621 ttattgcaag aagttgttga ccgcgatgct cttgtcgcta ccaagaaaag ccgtaaggag
     1681 cgtcaaaaat tggaacagac tgttttgcga tctgctgctg ttgatgaaag taatagagaa
     1741 tccttgcctt ctcaaatagt tgaaccagat gaagtggaat ctacagaaac ggttcactct
     1801 caatacgaag acattgaggt gtgggactat gaacaacttc gtgaagaata tgggttttaa
     1861 acaatgacag aagctcaggc gatcgccaag cagttgggtg gggtaaaacc ggatgatgag
     1921 tggttacaag ctgaaattgc tcgtctcaag ggtaagagca ttgtgccttt acagcaggta
     1981 aaaactctcc atgattggtt agatggcaag cgcaaggcaa gaaaatcttg ccgagtagtt
     2041 ggggaatcga gaactggcaa gacagttgct tgtgatgcct acagatacag gcacaaacct
     2101 cagcaggaag ctggacgacc tccaactgtg cctgtcgttt atattcgacc tcaccaaaaa
     2161 tgtggcccca aggatttgtt taaaaagatt actgagtacc tcaagtatcg ggtaacaaaa
     2221 gggactgtat ctgattttcg agataggacg atagaagtac tcaagggttg tggcgtagag
     2281 atgctaatta ttgatgaagc tgaccgtctc aagcctgaaa cttttgctga tgtgcgagat
     2341 attgccgaag atttaggaat tgctgtggta ctggtaggaa cagaccgttt ggatgcggta
     2401 attaagcggg atgagcaggt tctcgaacgc tttcgggcgc atcttcgctt tggtaaattg
     2461 tcgggagagg attttaagaa caccgtagaa atgtgggaac aaatggtttt gaaactgcca
     2521 gtatcttcta atctaaagag caaggagatg ctacggattc tcacgtcagc aactgaaggc
     2581 tacattggtc gccttgatga gattcttagg gaagctgcaa ttcgttcctt atcaagagga
     2641 ttgaagaaga ttgacaaggc tgttttacag gaagtagcta aggagtacaa atgactgcag
     2701 tgataaaacc agaaaaaggt gtgaaattaa ctaagtccct gaattgatct ggttgtccaa
     2761 aaaatttgtg cgatcgcatg gcaagattat tcctactatt gatgtggcgt gagtcggata
     2821 acttgctcta atgggatccc ggttaatacc aatagatcag atatgagtaa taaacattac
     2881 gacctttacc tgtggtattc actgttggca cgacaacacc tttatcgcgc caatactgta
     2941 gctgacgacg cgaacaatta gtgatttgag ccgcttctgt gcttgtgtag aattttcctt
     3001 ccatagaaat catcctacag aagtagtttt ttaaatttac attttttaaa agcacattat
     3061 gtattcatgt gatattattt tttcatgagt caaataacta ttcaagctcg acttatttcc
     3121 tttgaatcaa accgccaaca actctggaag ttgatggcag atttaaacac gccgttaatt
     3181 aacgaactgc tttgccagtt aggtcaacac cccgacttcg agaagtggca acaaaagggt
     3241 aaactcccgt ctaccgttgt gagccagtta tgtcaacctc tcaaaactga ccctcgcttt
     3301 gcaggtcagc ccagccgttt atatatgtcg gcaattcata ttgtggacta catctacaag
     3361 tcctggctgg ctatacagaa acggcttcaa cagcagctag atggaaagac gcgctggcta
     3421 gaaatgctca atagcgatgc tgaattagta gaacttagtg gtgacacttt agaggctatt
     3481 cgtgtcaaag ctgctgaaat tttggcaata gctatgccag catctgagtc agatagcgct
     3541 tcacctaaag ggaaaaaagg taaaaaggag aaaaaaccct catcttctag ccctaagcgt
     3601 agtttatcca agacattatt tgacgcttac caagaaacgg aagatatcaa gagccgtagc
     3661 gccatcagct acctgttaaa aaatggctgc aaacttactg acaaagaaga agattcagaa
     3721 aaatttgcta aacgtcgtcg tcaagttgaa atccaaattc aaaggcttac cgaaaagtta
     3781 ataagtcgga tgcctaaagg tcgagatttg accaatgcta aatggttgga gacactcttg
     3841 actgctacaa ccactgttgc tgaagacaac gcccaagcca aacgctggca ggatattctg
     3901 ttaactcgat caagttctct cccattcccc cttgtttttg aaaccaacga ggatatggtt
     3961 tggtcaaaga atcaaaaggg taggctgtgt gttcacttca atggcttaag cgatttaatt
     4021 tttgaggtgt actgcggcaa tcgtcaactt cactggtttc aacgcttcct agaagaccaa
     4081 cagactaaac gcaaaagcaa aaatcagcat tctagcggct tgttcacact cagaaatggt
     4141 catctagttt ggcttgaagg tgagggtaaa ggggaacctt ggaatcttca ccacttgacc
     4201 ctttactgct gtgttgacaa tcgcttgtgg acagaggagg gaacagaaat cgttcgccaa
     4261 gagaaagcag atgaaattac taaattcatc acaaacatga agaagaaaag cgatctaagc
     4321 gatacacagc aagctttgat tcaacgtaaa caatcaacac ttactcgaat aaacaattcc
     4381 tttgagcgtc ctagccaacc cctttatcaa ggtcaatcac acattttggt tggagtaagc
     4441 ctgggactag aaaaacctgc cacagtagca gtagtagatg cgatcgccaa caaagtcttg
     4501 gcttaccgga gtattaaaca attacttggc gacaattacg aactgctaaa tcgccagaga
     4561 cgacaacagc agtacctatc tcacgaacgc cacaaagcac aaaaaaactt ctctcccaat
     4621 caatttggag catctgagtt agggcaacat atagacagat tattagctaa agcaattgta
     4681 gcgttagcga gaacctacaa agctggcagt attgtcttgc ccaagttagg ggatatgcgg
     4741 gaggttgtcc aaagtgaaat tcaagctata gcagaacaaa aatttcccgg ttatattgaa
     4801 ggtcagcaaa aatatgccaa acagtaccgg gttaatgttc atcggtggag ctacggcaga
     4861 ttaattcaaa gcattcaaag taaagcagct caaacaggaa ttgtgattga ggagggaaaa
     4921 caacctattc gaggtagtcc ccacgacaaa gcaaaggaat tagcactttc tgcttacaat
     4981 ctccgcctaa ctaggcgaag tagcggcagc gagactcccg ggacctcaga gtccgccaca
     5041 cccgaaagtg ggagcataga agcaccagat gttaaacctt ggctattctt gattaaaccc
     5101 tatgaagggg aaagcctgag ccactttctt ggcaggttca gacgtgccaa ccatttatcc
     5161 gcaagtggat tgggtacttt ggcaggaatt ggtgctatag tggcacgttg ggaaagattt
     5221 cattttaatc ctcgccctag tcagcaagaa ttggaagcga tcgcatctgt agtagaagtg
     5281 gatgctcaaa ggttagccca gatgttaccg cctgctggag tgggaatgca gcatgagcca
     5341 attcgcttgt gtggggcttg ttatgccgag tcgccttgtc accgaattga atggcagtac
     5401 aagtcggtgt ggaagtgcga tcgccatcaa ctcaagattt tagcaaagtg tccaaactgt
     5461 caagcacctt ttaaaatgcc tgcgctgtgg gaggatgggt gctgtcacag atgtaggatg
     5521 ccgtttgcag aaatggcaaa gctacagaag gtttgagtaa ggatctccag gcatcaaata
     5581 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     5641 gctctctact agagagaccc tgacagctag ctcagtccta ggtataatgc tagcatatta
     5701 atagcgccgc aattcatgct gcttgcagcc tctgaatttt gttaaatgag ggttagtttg
     5761 actgtataaa tacagtcttg ctttctgacc ctggtagctg ctcaccctga tgctgctgtc
     5821 aatagacagg ataggtgcgc tcccagcaat aagggcgcgg atgtactgct gtagtggcta
     5881 ctgaatcacc cccgatcaag ggggaaccct aaatgggttg aaaggaagag cgagatctcg
     5941 agctcttcgg ctcaccttcg ggtgggcctt tctgcgcagg tggcactttt cggggaaatg
     6001 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6061 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6121 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6181 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6241 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     6301 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     6361 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     6421 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     6481 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6541 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6601 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6661 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6721 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6781 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6841 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6901 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6961 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     7021 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7081 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     7141 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     7201 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     7261 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     7321 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     7381 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     7441 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     7501 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     7561 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7621 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7681 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7741 cggccttttt acggttcctg gccttttgct
//
