LOCUS       sequence-354678-        8528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Y2-ShHELIX containing a nicking Y2 I-AniI fusion to 
            ShTnsB. 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 8528)
  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 8528)
  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: pY2ShHELIX_sgRNA_entry (CJT30)
FEATURES             Location/Qualifiers
     source          1..8528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(28..33,34..51,52..58)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             59..70
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      6304..6375
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        6389..6423
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      6678..6705
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        6732..6836
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6837..6905,6906..7697)
                     /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(7055..7074)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7868..8456
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8357..8376
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgatctcaag aagatcatct tattaatttt acactttatg cttccggctc gtatgttgaa
       61 agaggagaaa ggatctatgg gcagcgattt aacatacgca tatctagtag gcctgtacga
      121 gggtgacggt tatttctcta tcaccaagaa aggtaaatac ttgacgtatg aactgggcat
      181 cgagctgtcg attaaggatg ttcagctgat ctacaagatc aaaaaaatct tgggcattgg
      241 tattgttagc ttcagaaagc gcaatgaaat tgagatggtt gcactgcgta ttcgtgacaa
      301 aaaccacctg aaaagcaaaa tcctgccgat ttttgaaaag tatccgatgt tttccaacaa
      361 acaatatgat tacttgcgct tccgcaacgc gctgctgagc ggtattattt acttggaaga
      421 tctgccagac tacacccgtt ctgatgaacc gctgaatagc attgagagca tcatcaatac
      481 ctcctatttt agcgcgtggc tggtcggttt tatcgaggcc gagggctgct tttccgtgta
      541 taaacttaac aaggacgatg attacttaat cgctagcttc gacatcgcgc agcgtgacgg
      601 cgacatcctg atttcagcga ttcgcaagta cctgagcttc actacgaaag tgtacctcga
      661 caagaccaat tgttcgaaat tgaaggttac ctccgtgcgt agcgtggaaa acatcatcaa
      721 gttcctgcaa aacgctccgg tcaaactgtt gggaaacatg aaattacaat ataagctgtg
      781 gttgaagcag ctgcgtaaga ttagccgtta tagcgagaaa atcaagatac cgtctaacta
      841 cagcggcagc gagactcccg ggacctcaga gtccgccaca cccgaaagtg ggagcaacag
      901 tcagcaaaat cctgatttag ctgttcatcc cttggcaatt cctatggaag gcttactagg
      961 agaaagtgct acaactcttg agaagaatgt aattgccaca caactctcag aggaagccca
     1021 agtaaagcta gaggtaatcc aaagtttact ggaaccctgc gatcgcacaa cttatgggca
     1081 aaagttgcgg gaagcagcag agaaactaaa tgtatcgttg cgaacggtac aaaggttggt
     1141 gaaaaactgg gaacaagatg gcttagtcgg actcactcaa acaagtaggg ctgataaagg
     1201 aaaacaccgc attggtgagt tttgggaaaa cttcattacc aaaacctaca aggagggtaa
     1261 caagggaagt aaacgtatga cccctaaaca agttgctctc agagtcgagg ctaaagcccg
     1321 tgaattaaaa gactctaagc cgcccaatta caaaaccgtg ttacgggtat tagcacccat
     1381 tttggaaaag caacaaaaag ccaagagtat ccgcagtcct ggttggagag gaactacgct
     1441 ttcggttaaa acccgtgaag gaaaagattt atcggttgat tacagtaacc atgtttggca
     1501 atgtgaccat acccgcgtgg atgtgttgct ggtagatcaa catggtgaaa ttttaagtcg
     1561 tccctggcta acaacagtaa ttgatactta ctctcgttgc attatgggta tcaacttggg
     1621 ctttgatgca cccagttctg gggtagtagc attagcgtta cgccatgcaa ttctaccaaa
     1681 gcgttacggt tccgagtaca aactgcattg tgagtgggga acctatggaa aaccagaaca
     1741 tttttatact gatggcggta aagactttcg ctctaaccac ttgagtcaga ttggggcgca
     1801 attgggattt gtctgtcatt tacgcgatcg cccttctgaa ggtggagtag tagaacgtcc
     1861 cttcaaaaca ttaaatgacc aactattttc aacgcttcct gggtacaccg gatctaatgt
     1921 gcaggaacgc ccagaagatg cagagaagga cgcaagactt actttgcgag aactagaaca
     1981 gttacttgtg cgttacatcg tagatcgtta caaccaaagt attgatgcgc ggatgggcga
     2041 ccaaacgcgc tttgagcgtt gggaagcagg attgcctaca gtgccagtac caataccaga
     2101 acgagatttg gatatttgtt taatgaagca gtcacggcgc actgtgcaaa gaggtggttg
     2161 tttgcagttt cagaatttaa tgtatcgggg ggaatatttg gcaggttatg ccggagaaac
     2221 tgtcaactta aggtttgacc ccagagacat tacaacaatt ttggtttatc gccaggaaaa
     2281 caatcaggaa gtatttctga ctcgcgctca cgctcaaggt ttggagacag agcaactggc
     2341 attagatgag gctgaggcag caagtcgcag actccgtacc gcagggaaaa ctatcagtaa
     2401 ccaatcatta ttgcaagaag ttgttgaccg cgatgctctt gtcgctacca agaaaagccg
     2461 taaggagcgt caaaaattgg aacagactgt tttgcgatct gctgctgttg atgaaagtaa
     2521 tagagaatcc ttgccttctc aaatagttga accagatgaa gtggaatcta cagaaacggt
     2581 tcactctcaa tacgaagaca ttgaggtgtg ggactatgaa caacttcgtg aagaatatgg
     2641 gttttaaaca atgacagaag ctcaggcgat cgccaagcag ttgggtgggg taaaaccgga
     2701 tgatgagtgg ttacaagctg aaattgctcg tctcaagggt aagagcattg tgcctttaca
     2761 gcaggtaaaa actctccatg attggttaga tggcaagcgc aaggcaagaa aatcttgccg
     2821 agtagttggg gaatcgagaa ctggcaagac agttgcttgt gatgcctaca gatacaggca
     2881 caaacctcag caggaagctg gacgacctcc aactgtgcct gtcgtttata ttcgacctca
     2941 ccaaaaatgt ggccccaagg atttgtttaa aaagattact gagtacctca agtatcgggt
     3001 aacaaaaggg actgtatctg attttcgaga taggacgata gaagtactca agggttgtgg
     3061 cgtagagatg ctaattattg atgaagctga ccgtctcaag cctgaaactt ttgctgatgt
     3121 gcgagatatt gccgaagatt taggaattgc tgtggtactg gtaggaacag accgtttgga
     3181 tgcggtaatt aagcgggatg agcaggttct cgaacgcttt cgggcgcatc ttcgctttgg
     3241 taaattgtcg ggagaggatt ttaagaacac cgtagaaatg tgggaacaaa tggttttgaa
     3301 actgccagta tcttctaatc taaagagcaa ggagatgcta cggattctca cgtcagcaac
     3361 tgaaggctac attggtcgcc ttgatgagat tcttagggaa gctgcaattc gttccttatc
     3421 aagaggattg aagaagattg acaaggctgt tttacaggaa gtagctaagg agtacaaatg
     3481 atagaagcac cagatgttaa accttggcta ttcttgatta aaccctatga aggggaaagc
     3541 ctgagccact ttcttggcag gttcagacgt gccaaccatt tatccgcaag tggattgggt
     3601 actttggcag gaattggtgc tatagtggca cgttgggaaa gatttcattt taatcctcgc
     3661 cctagtcagc aagaattgga agcgatcgca tctgtagtag aagtggatgc tcaaaggtta
     3721 gcccagatgt taccgcctgc tggagtggga atgcagcatg agccaattcg cttgtgtggg
     3781 gcttgttatg ccgagtcgcc ttgtcaccga attgaatggc agtacaagtc ggtgtggaag
     3841 tgcgatcgcc atcaactcaa gattttagca aagtgtccaa actgtcaagc accttttaaa
     3901 atgcctgcgc tgtgggagga tgggtgctgt cacagatgta ggatgccgtt tgcagaaatg
     3961 gcaaagctac agaaggtttg atgataaaac cagaaaaagg tgtgaaatta actaagtccc
     4021 tgaattgatc tggttgtcca aaaaatttgt gcgatcgcat ggcaagatta ttcctactat
     4081 tgatgtggcg tgagtcggat aacttgctct aatgggatcc cggttaatac caatagatca
     4141 gatatgagta ataaacatta cgacctttac ctgtggtatt cactgttggc acgacaacac
     4201 ctttatcgcg ccaatactgt agctgacgac gcgaacaatt agtgatttga gccgcttctg
     4261 tgcttgtgta gaattttcct tccatagaaa tcatcctaca gaagtagttt tttaaattta
     4321 cattttttaa aagcacatta tgtattcatg tgatattatt tttttatgag tcaaataact
     4381 attcaagctc gacttatttc ctttgaatca aaccgccaac aactctggaa gttgatggca
     4441 gatttaaaca cgccgttaat taacgaactg ctttgccagt taggtcaaca ccccgacttc
     4501 gagaagtggc aacaaaaggg taaactcccg tctaccgttg tgagccagtt atgtcaacct
     4561 ctcaaaactg accctcgctt tgcaggtcag cccagccgtt tatatatgtc ggcaattcat
     4621 attgtggact acatctacaa gtcctggctg gctatacaga aacggcttca acagcagcta
     4681 gatggaaaga cgcgctggct agaaatgctc aatagcgatg ctgaattagt agaacttagt
     4741 ggtgacactt tagaggctat tcgtgtcaaa gctgctgaaa ttttggcaat agctatgcca
     4801 gcatctgagt cagatagcgc ttcacctaaa gggaaaaaag gtaaaaagga gaaaaaaccc
     4861 tcatcttcta gccctaagcg tagtttatcc aagacattat ttgacgctta ccaagaaacg
     4921 gaagatatca agagccgtag cgccatcagc tacctgttaa aaaatggctg caaacttact
     4981 gacaaagaag aagattcaga aaaatttgct aaacgtcgtc gtcaagttga aatccaaatt
     5041 caaaggctta ccgaaaagtt aataagtcgg atgcctaaag gtcgagattt gaccaatgct
     5101 aaatggttgg agacactctt gactgctaca accactgttg ctgaagacaa cgcccaagcc
     5161 aaacgctggc aggatattct gttaactcga tcaagttctc tcccattccc ccttgttttt
     5221 gaaaccaacg aggatatggt ttggtcaaag aatcaaaagg gtaggctgtg tgttcacttc
     5281 aatggcttaa gcgatttaat ttttgaggtg tactgcggca atcgtcaact tcactggttt
     5341 caacgcttcc tagaagacca acagactaaa cgcaaaagca aaaatcagca ttctagcggc
     5401 ttgttcacac tcagaaatgg tcatctagtt tggcttgaag gtgagggtaa aggggaacct
     5461 tggaatcttc accacttgac cctttactgc tgtgttgaca atcgcttgtg gacagaggag
     5521 ggaacagaaa tcgttcgcca agagaaagca gatgaaatta ctaaattcat cacaaacatg
     5581 aagaagaaaa gcgatctaag cgatacacag caagctttga ttcaacgtaa acaatcaaca
     5641 cttactcgaa taaacaattc ctttgagcgt cctagccaac ccctttatca aggtcaatca
     5701 cacattttgg ttggagtaag cctgggacta gaaaaacctg ccacagtagc agtagtagat
     5761 gcgatcgcca acaaagtctt ggcttaccgg agtattaaac aattacttgg cgacaattac
     5821 gaactgctaa atcgccagag acgacaacag cagtacctat ctcacgaacg ccacaaagca
     5881 caaaaaaact tctctcccaa tcaatttgga gcatctgagt tagggcaaca tatagacaga
     5941 ttattagcta aagcaattgt agcgttagcg agaacctaca aagctggcag tattgtcttg
     6001 cccaagttag gggatatgcg ggaggttgtc caaagtgaaa ttcaagctat agcagaacaa
     6061 aaatttcccg gttatattga aggtcagcaa aaatatgcca aacagtaccg ggttaatgtt
     6121 catcggtgga gctacggcag attaattcaa agcattcaaa gtaaagcagc tcaaacagga
     6181 attgtgattg aggagggaaa acaacctatt cgaggtagtc cccacgacaa agcaaaggaa
     6241 ttagcacttt ctgcttacaa tctccgccta actaggcgaa gttaagtaag gatctccagg
     6301 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     6361 tcggtgaacg ctctctacta gagagaccct gacagctagc tcagtcctag gtataatgct
     6421 agcatattaa tagcgccgca attcatgctg cttgcagcct ctgaattttg ttaaatgagg
     6481 gttagtttga ctgtataaat acagtcttgc tttctgaccc tggtagctgc tcaccctgat
     6541 gctgctgtca atagacagga taggtgcgct cccagcaata agggcgcgga tgtactgctg
     6601 tagtggctac tgaatcaccc ccgatcaagg gggaacccta aatgggttga aaggaagagc
     6661 gagatctcga gctcttcggc tcaccttcgg gtgggccttt ctgcgcaggt ggcacttttc
     6721 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6781 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6841 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6901 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6961 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     7021 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     7081 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     7141 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     7201 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     7261 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     7321 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     7381 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     7441 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     7501 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     7561 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     7621 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     7681 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     7741 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     7801 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     7861 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     7921 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     7981 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     8041 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     8101 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     8161 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     8221 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     8281 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     8341 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     8401 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     8461 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct caacgccaag
     8521 ctccaaaa
//
