LOCUS       sequence-354816-        7764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3-component ShCAST containing a Cas12k-TnsC 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 7764)
  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 7764)
  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-TnsC)-sgRNA_entry (CJT28)
FEATURES             Location/Qualifiers
     source          1..7764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     355..374
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(554..559,560..577,578..584)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             585..596
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      6066..6137
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        6151..6185
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      6440..6467
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        6494..6598
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6599..6667,6668..7459)
                     /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(6817..6836)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7630..7764,1..454)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
       61 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      121 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      181 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      241 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      301 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      361 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      421 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      481 gttcctggcc ttttgctggc cttttgctca acgccaagct ccaaaacgat ctcaagaaga
      541 tcatcttatt aattttacac tttatgcttc cggctcgtat gttgaaagag gagaaaggat
      601 ctatgaacag tcagcaaaat cctgatttag ctgttcatcc cttggcaatt cctatggaag
      661 gcttactagg agaaagtgct acaactcttg agaagaatgt aattgccaca caactctcag
      721 aggaagccca agtaaagcta gaggtaatcc aaagtttact ggaaccctgc gatcgcacaa
      781 cttatgggca aaagttgcgg gaagcagcag agaaactaaa tgtatcgttg cgaacggtac
      841 aaaggttggt gaaaaactgg gaacaagatg gcttagtcgg actcactcaa acaagtaggg
      901 ctgataaagg aaaacaccgc attggtgagt tttgggaaaa cttcattacc aaaacctaca
      961 aggagggtaa caagggaagt aaacgtatga cccctaaaca agttgctctc agagtcgagg
     1021 ctaaagcccg tgaattaaaa gactctaagc cgcccaatta caaaaccgtg ttacgggtat
     1081 tagcacccat tttggaaaag caacaaaaag ccaagagtat ccgcagtcct ggttggagag
     1141 gaactacgct ttcggttaaa acccgtgaag gaaaagattt atcggttgat tacagtaacc
     1201 atgtttggca atgtgaccat acccgcgtgg atgtgttgct ggtagatcaa catggtgaaa
     1261 ttttaagtcg tccctggcta acaacagtaa ttgatactta ctctcgttgc attatgggta
     1321 tcaacttggg ctttgatgca cccagttctg gggtagtagc attagcgtta cgccatgcaa
     1381 ttctaccaaa gcgttacggt tccgagtaca aactgcattg tgagtgggga acctatggaa
     1441 aaccagaaca tttttatact gatggcggta aagactttcg ctctaaccac ttgagtcaga
     1501 ttggggcgca attgggattt gtctgtcatt tacgcgatcg cccttctgaa ggtggagtag
     1561 tagaacgtcc cttcaaaaca ttaaatgacc aactattttc aacgcttcct gggtacaccg
     1621 gatctaatgt gcaggaacgc ccagaagatg cagagaagga cgcaagactt actttgcgag
     1681 aactagaaca gttacttgtg cgttacatcg tagatcgtta caaccaaagt attgatgcgc
     1741 ggatgggcga ccaaacgcgc tttgagcgtt gggaagcagg attgcctaca gtgccagtac
     1801 caataccaga acgagatttg gatatttgtt taatgaagca gtcacggcgc actgtgcaaa
     1861 gaggtggttg tttgcagttt cagaatttaa tgtatcgggg ggaatatttg gcaggttatg
     1921 ccggagaaac tgtcaactta aggtttgacc ccagagacat tacaacaatt ttggtttatc
     1981 gccaggaaaa caatcaggaa gtatttctga ctcgcgctca cgctcaaggt ttggagacag
     2041 agcaactggc attagatgag gctgaggcag caagtcgcag actccgtacc gcagggaaaa
     2101 ctatcagtaa ccaatcatta ttgcaagaag ttgttgaccg cgatgctctt gtcgctacca
     2161 agaaaagccg taaggagcgt caaaaattgg aacagactgt tttgcgatct gctgctgttg
     2221 atgaaagtaa tagagaatcc ttgccttctc aaatagttga accagatgaa gtggaatcta
     2281 cagaaacggt tcactctcaa tacgaagaca ttgaggtgtg ggactatgaa caacttcgtg
     2341 aagaatatgg gttttaaaca atgatagaag caccagatgt taaaccttgg ctattcttga
     2401 ttaaacccta tgaaggggaa agcctgagcc actttcttgg caggttcaga cgtgccaacc
     2461 atttatccgc aagtggattg ggtactttgg caggaattgg tgctatagtg gcacgttggg
     2521 aaagatttca ttttaatcct cgccctagtc agcaagaatt ggaagcgatc gcatctgtag
     2581 tagaagtgga tgctcaaagg ttagcccaga tgttaccgcc tgctggagtg ggaatgcagc
     2641 atgagccaat tcgcttgtgt ggggcttgtt atgccgagtc gccttgtcac cgaattgaat
     2701 ggcagtacaa gtcggtgtgg aagtgcgatc gccatcaact caagatttta gcaaagtgtc
     2761 caaactgtca agcacctttt aaaatgcctg cgctgtggga ggatgggtgc tgtcacagat
     2821 gtaggatgcc gtttgcagaa atggcaaagc tacagaaggt ttgatgataa aaccagaaaa
     2881 aggtgtgaaa ttaactaagt ccctgaattg atctggttgt ccaaaaaatt tgtgcgatcg
     2941 catggcaaga ttattcctac tattgatgtg gcgtgagtcg gataacttgc tctaatggga
     3001 tcccggttaa taccaataga tcagatatga gtaataaaca ttacgacctt tacctgtggt
     3061 attcactgtt ggcacgacaa cacctttatc gcgccaatac tgtagctgac gacgcgaaca
     3121 attagtgatt tgagccgctt ctgtgcttgt gtagaatttt ccttccatag aaatcatcct
     3181 acagaagtag ttttttaaat ttacattttt taaaagcaca ttatgtattc atgtgatatt
     3241 atttttttat gagtcaaata actattcaag ctcgacttat ttcctttgaa tcaaaccgcc
     3301 aacaactctg gaagttgatg gcagatttaa acacgccgtt aattaacgaa ctgctttgcc
     3361 agttaggtca acaccccgac ttcgagaagt ggcaacaaaa gggtaaactc ccgtctaccg
     3421 ttgtgagcca gttatgtcaa cctctcaaaa ctgaccctcg ctttgcaggt cagcccagcc
     3481 gtttatatat gtcggcaatt catattgtgg actacatcta caagtcctgg ctggctatac
     3541 agaaacggct tcaacagcag ctagatggaa agacgcgctg gctagaaatg ctcaatagcg
     3601 atgctgaatt agtagaactt agtggtgaca ctttagaggc tattcgtgtc aaagctgctg
     3661 aaattttggc aatagctatg ccagcatctg agtcagatag cgcttcacct aaagggaaaa
     3721 aaggtaaaaa ggagaaaaaa ccctcatctt ctagccctaa gcgtagttta tccaagacat
     3781 tatttgacgc ttaccaagaa acggaagata tcaagagccg tagcgccatc agctacctgt
     3841 taaaaaatgg ctgcaaactt actgacaaag aagaagattc agaaaaattt gctaaacgtc
     3901 gtcgtcaagt tgaaatccaa attcaaaggc ttaccgaaaa gttaataagt cggatgccta
     3961 aaggtcgaga tttgaccaat gctaaatggt tggagacact cttgactgct acaaccactg
     4021 ttgctgaaga caacgcccaa gccaaacgct ggcaggatat tctgttaact cgatcaagtt
     4081 ctctcccatt cccccttgtt tttgaaacca acgaggatat ggtttggtca aagaatcaaa
     4141 agggtaggct gtgtgttcac ttcaatggct taagcgattt aatttttgag gtgtactgcg
     4201 gcaatcgtca acttcactgg tttcaacgct tcctagaaga ccaacagact aaacgcaaaa
     4261 gcaaaaatca gcattctagc ggcttgttca cactcagaaa tggtcatcta gtttggcttg
     4321 aaggtgaggg taaaggggaa ccttggaatc ttcaccactt gaccctttac tgctgtgttg
     4381 acaatcgctt gtggacagag gagggaacag aaatcgttcg ccaagagaaa gcagatgaaa
     4441 ttactaaatt catcacaaac atgaagaaga aaagcgatct aagcgataca cagcaagctt
     4501 tgattcaacg taaacaatca acacttactc gaataaacaa ttcctttgag cgtcctagcc
     4561 aaccccttta tcaaggtcaa tcacacattt tggttggagt aagcctggga ctagaaaaac
     4621 ctgccacagt agcagtagta gatgcgatcg ccaacaaagt cttggcttac cggagtatta
     4681 aacaattact tggcgacaat tacgaactgc taaatcgcca gagacgacaa cagcagtacc
     4741 tatctcacga acgccacaaa gcacaaaaaa acttctctcc caatcaattt ggagcatctg
     4801 agttagggca acatatagac agattattag ctaaagcaat tgtagcgtta gcgagaacct
     4861 acaaagctgg cagtattgtc ttgcccaagt taggggatat gcgggaggtt gtccaaagtg
     4921 aaattcaagc tatagcagaa caaaaatttc ccggttatat tgaaggtcag caaaaatatg
     4981 ccaaacagta ccgggttaat gttcatcggt ggagctacgg cagattaatt caaagcattc
     5041 aaagtaaagc agctcaaaca ggaattgtga ttgaggaggg aaaacaacct attcgaggta
     5101 gtccccacga caaagcaaag gaattagcac tttctgctta caatctccgc ctaactaggc
     5161 gaagtagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa agtgggagca
     5221 cagaagctca ggcgatcgcc aagcagttgg gtggggtaaa accggatgat gagtggttac
     5281 aagctgaaat tgctcgtctc aagggtaaga gcattgtgcc tttacagcag gtaaaaactc
     5341 tccatgattg gttagatggc aagcgcaagg caagaaaatc ttgccgagta gttggggaat
     5401 cgagaactgg caagacagtt gcttgtgatg cctacagata caggcacaaa cctcagcagg
     5461 aagctggacg acctccaact gtgcctgtcg tttatattcg acctcaccaa aaatgtggcc
     5521 ccaaggattt gtttaaaaag attactgagt acctcaagta tcgggtaaca aaagggactg
     5581 tatctgattt tcgagatagg acgatagaag tactcaaggg ttgtggcgta gagatgctaa
     5641 ttattgatga agctgaccgt ctcaagcctg aaacttttgc tgatgtgcga gatattgccg
     5701 aagatttagg aattgctgtg gtactggtag gaacagaccg tttggatgcg gtaattaagc
     5761 gggatgagca ggttctcgaa cgctttcggg cgcatcttcg ctttggtaaa ttgtcgggag
     5821 aggattttaa gaacaccgta gaaatgtggg aacaaatggt tttgaaactg ccagtatctt
     5881 ctaatctaaa gagcaaggag atgctacgga ttctcacgtc agcaactgaa ggctacattg
     5941 gtcgccttga tgagattctt agggaagctg caattcgttc cttatcaaga ggattgaaga
     6001 agattgacaa ggctgtttta caggaagtag ctaaggagta caaataagta aggatctcca
     6061 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     6121 tgtcggtgaa cgctctctac tagagagacc ctgacagcta gctcagtcct aggtataatg
     6181 ctagcatatt aatagcgccg caattcatgc tgcttgcagc ctctgaattt tgttaaatga
     6241 gggttagttt gactgtataa atacagtctt gctttctgac cctggtagct gctcaccctg
     6301 atgctgctgt caatagacag gataggtgcg ctcccagcaa taagggcgcg gatgtactgc
     6361 tgtagtggct actgaatcac ccccgatcaa gggggaaccc taaatgggtt gaaaggaaga
     6421 gcgagatctc gagctcttcg gctcaccttc gggtgggcct ttctgcgcag gtggcacttt
     6481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     6541 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     6601 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     6661 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     6721 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     6781 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     6841 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     6901 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     6961 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     7021 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     7081 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     7141 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     7201 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     7261 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     7321 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     7381 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     7441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     7501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     7561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     7621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     7681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7741 aactggcttc agcagagcgc agat
//
