LOCUS       sequence-358225-        8032 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ShoCAST. 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 8032)
  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 8032)
  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: pShoCAST-sgRNA_entry (BO1)
FEATURES             Location/Qualifiers
     source          1..8032
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     411..430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(610..615,616..633,634..640)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             641..652
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      6269..6340
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        6354..6388
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      6764..6791
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        6818..6922
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6923..6991,6992..7783)
                     /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(7141..7160)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7954..8032,1..510)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
       61 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
      121 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
      181 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
      241 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
      301 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
      361 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
      421 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      481 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      541 ctggcctttt gctggccttt tgctcaacgc caagctccaa aacgatctca agaagatcat
      601 cttattaatt ttacacttta tgcttccggc tcgtatgttg aaagaggaga aaggatctat
      661 gggcttagac gaagaatttg agttcactga ggaattgacg caagctccag atgttattgt
      721 gcttgacaag agtcattttg tggtagaccc atcccaaatt attctgcaaa catcggataa
      781 gcataaactg agatttaatc ttatcaagtg gttcgctgaa tcccccaaca ttactattaa
      841 atctcagaga aaacaggcag tcgtagatac ccttggtgtt tctactcggc aggtggagcg
      901 tctcctcaag caatatcata atggtgagtt atcagagaca gcgggagtac aacgctcaga
      961 taaggggaaa cttcgcatca gccaatactg ggaagattat attaaaacaa cttatgaaaa
     1021 aagtctgaaa gataaacatc caatgttgcc agcagctgtc gtgcgggaag tgaagcgaca
     1081 cgcgatcgtt gacctcggac tgaaaccagg agattatccc catccagcca ccatctatag
     1141 aaatttagct cctttaatcg agcaacacac gcggaagaaa aaagttagaa atccgggttc
     1201 gggatcttgg ctgaccgttg tcacgcgaga tggtcagcta ctgaaggctg actttagtaa
     1261 ccaaattatc caatgcgacc ataccgagct ggatattcat atagttgata gtcatggcag
     1321 tttattgtct gaccgtcctt ggctaactac tgttgtcgat acttattcaa gttgcattct
     1381 tggttttcat ttatggatca aacaaccagg ttctacggag gtagctcttg ccttgagaca
     1441 cgccattcta cccaaaaatt atccagagga ctacaaactt ggcaaagttt gggaaatata
     1501 tggacctcct tttcagtatt tttttactga tggtggaaaa gattttaatt caaagcatct
     1561 aaaagccatt ggtaagaaat taggatttca gtgcgaacta cgcaatcgcc cgccccaagg
     1621 tggaattgta gaacgtcttt ttaaaaccat taatactcaa gttcttaaag agcttcccgg
     1681 ttacacgggg gcaaacgttc aagaacgccc gaaaaatgca gagaaagaag cctgtttaac
     1741 aatacaggat ttggataaaa ttctagccag cttcttttgt gatatttata accacgaacc
     1801 ctatcccaag gagcctcgca acacgagatt tgaacgctgg tttaaaggta tggggggaaa
     1861 attacccgaa cctttggacg agcgagaatt ggatatttgt ttgatgaaag aagcacaacg
     1921 agttgttcaa gctcatggta gcatacagtt tgaaaaccta atttatcgag gagaagctct
     1981 gaaggcatat agaggtgaat acgtcaccct aagatacgat cccgaccatg tcctgacttt
     2041 atatgtctat agttgcgaag ctgatgataa tgcagaagag ttcttgggct atgctcacgc
     2101 tataaacatg gatactcatg acttgagtat agaagaattg aaaactctca ataaagaacg
     2161 aagtaaagct cgtagcgatc attacaacta tgatgcttta ttagcattgg gtaaacgcaa
     2221 agaacttgta gaagaaagaa aacaagacaa gaaagcaaaa aggcaatcag agcaaaaacg
     2281 tctccgcact gcttccaaga aaaactcaaa tgttattgaa ctacgcaaaa gtagagcttc
     2341 gagttcttca agtaaagatg accgacagga aattttacca gagagagttt ctagggacga
     2401 actaaagccg gagaaaacag aactgaaata tgaggaaaat ctgttagcac agaccgatac
     2461 tcaaaaacag gagagacata agttagttgt ctctgaccgt aaaaaaaatt taaagaacat
     2521 ttggtgataa aggaggtaaa taatggccat atctcaactg gcaacccaac catttgtcga
     2581 agtcttacct ccagagttag atagcaaagc tcaaattgct aaaactattg atattgagga
     2641 gcttttcaga attaatttta tcacaaccga tcggtcatca gaatgtttca gatggttaga
     2701 tgagctgcgt atcctgaaac aatgcggtcg gatcattgga ccaagaaatg taggcaaaag
     2761 ccgagcagtg cttcactatc gtaatgagga taaaaaacga gtttcttatg taaaggcttg
     2821 gtcggcatcg agttctaaac gtctattttc acaaatctta aaggatatta accatgctgc
     2881 atcaacaggt aaacgacagg atttacgtcc aagattagct ggtagtcttg aactatttgg
     2941 attagaattg gtcattgttg ataatgctga aaatcttcaa aaagaggcgc tcttagattt
     3001 aaagcaactt tttgaagaat gccatgttcc tattgtctta gttggaggta aggagttaga
     3061 tgatatttta gaagattttg atttgttgac taactttcca acgttatatg agtttgaacg
     3121 attggaacat gatgatttta taaaaacatt aaagacaatc gaattggata ttttatctct
     3181 tccagaagcg tctaagttaa gtgagggtaa catatttgca attttagctg agagtacagg
     3241 tgggaaaata ggaattttag tcaagatact aactaaagca gttttacatt ctcttaaaaa
     3301 gggatttggt aaagttgatg aaagcatttt agaaaaaatc gcaagtcgtt atggaacaaa
     3361 atacgttccc attgaaaaca aaaatagaaa cgattaatag aggaggatat aaatgattga
     3421 agatgatgaa attcgtctaa ggttgggcta tgttgaacca cacccaggtg aaagcatcag
     3481 tcattatttg ggacggcttc gtcgatttaa ggctaacagc cttccttcag gatatgctct
     3541 aggaaaaatt gcgggtcttg ggtctgttct gactcgatgg gaaaagttgt acttcaaccc
     3601 atttcccact caacaagaac tagaggcatt agcacaagtg attcaagtgg aggttgagaa
     3661 attaagagag atgctaccca ctaaaggtgt cacaatgatg cctcgaccca ttcgattatg
     3721 tgcggcttgc tatgcagagt caccttatca ccggatcgag tggcagttta aggacaaaat
     3781 gaagtgcgat cgccatcagt tgcgcttatt aacaaaatgc acaaactgtc agacaccatt
     3841 ccccattcct gctgattggg aaaaaggaga atgctcgcat tgttttttgt cttttgctaa
     3901 gatggtgaag tgtcaaaagc gtcgttagtg ataaaaccag aaaaaggtgt gaaattaact
     3961 aagtccctga attgatctgg ttgtccaaaa aatttgtgcg atcgcatggc aagattattc
     4021 ctactattga tgtggcgtga gtcggataac ttgctctaat gggatcccgg ttaataccaa
     4081 tagatcagat atgagtaata aacattacga cctttacctg tggtattcac tgttggcacg
     4141 acaacacctt tatcgcgcca atactgtagc tgacgacgcg aacaattagt gatttgagcc
     4201 gcttctgtgc ttgtgtagaa ttttccttcc atagaaatca tcctacagaa gtagtttttt
     4261 aaatttacat tttttaaaag cacattatgt attcatgtga tattattttt ttatgagcac
     4321 aatcacgatt caatgccgcc tagttgcaga agaagcgacc ctgcgctact tttgggaact
     4381 tatggctgag aagaatacgc cactcattaa tgaattgttg gagcagttag gacaacatcc
     4441 agattttgat acttgggtac aagcaggcaa gatgcctgaa aagacagttg agaacttgtg
     4501 caagtcccta gaagatcgag aaccttttgc aaatcagccc ggtcgttttc gtacttcggc
     4561 agttgctctg gtaaagtaca tttacaaatc ctggtttgcg ttacaaaaac gtcgggcgga
     4621 cagactagaa ggtaaagagc gctggttaaa gatgctcaaa agtgatgtgg aactggaacg
     4681 agaaagcaac tgtagtttgg atattattcg agctaaagct ggggaaattc ttgctaaagt
     4741 tactgagggg tgcgctccga gcaatcaaac atcctctaag agaaaaaaga agaaaactaa
     4801 aaaaagccaa gcgacaaaag atctccctac gcttttcgag attatattaa aggcttacga
     4861 acaagctgaa gaaagcctga cacgagctgc ccttgcttat ctccttaaaa atgactgtga
     4921 agtcagtgaa gttgatgaag attcagagaa atttaaaaag cgtcggcgta aaaaggaaat
     4981 agaaattgaa cgtttaagaa atcaactgaa aagtcggata cctaaaggtc gcgatttaac
     5041 cggagataag tggttgaaaa cgctagaaga agccacccga aatgttcctg aaaatgagga
     5101 tgaggcaaag gcttggcaag cccaactctt gagagaagct agttctgttc cttttcctgt
     5161 agcttatgaa actagtgaag atatgacttg gtttaccaac gagcaaggtc gaatctttgt
     5221 ttactttaat ggttcagcca aacataaatt tcaagtttat tgcgatcgcc gtcaacttca
     5281 ttggtttcaa cgttttgtag aggactttca aatcaagaaa aatggtgata aaaaaggtag
     5341 cgaaaaggag taccctgctg ggttattgac gctttgttcc actcgtctga gatggaaaga
     5401 aagcgcggaa aaaggagatc cttggaatgt tcatcgccta attctttctt gcactatcga
     5461 tacacgctta tggacacttg aaggaacgga acaggtgcga gctgaaaaga tcgctcaggt
     5521 tgaaaaaact attagcaagc gggagcaaga agttaatctg agtaagactc aacttgagcg
     5581 tttacaagcc aagcacagtg agagggagag attgaacaac atttttccca accgccctag
     5641 caagcccagt taccggggta aatctcatat tgcgatcggt gtaagtttca gtttggaaaa
     5701 cccagccact gttgccgtcg tagatgttgc aaccaagaaa gtccttactt accgcagttt
     5761 caaacaatta ctcggagata actacaacct tgcaaaccgg ctgcggcaac aaaagcaacg
     5821 tctttctcac gaacgtcaca aagctcagaa acaaggcgca ccaaattcat ttggagactc
     5881 agagttaggg cagtatgtag acagattgct tgcaaaatcg attgtcgcga tcgcaaaaac
     5941 ttatcaagct agtagtattg tcttgcctaa acttcgctat atgagggaaa tcatacataa
     6001 tgaagttcaa gctaaagccg aaaaaaagat tccaggatat aaagaaggtc aaaaacaata
     6061 tgccaaacag tatcggataa gcgttcatca atggagttat aatagattat cacagattct
     6121 tgaaagccaa gctacaaaag ctggaatttc tattgaacgt ggatcgcaag taatccaagg
     6181 tagctcccaa gaacaagcac gagatttagc tttatttgcc tataacgaac gccaacttag
     6241 tctgggttag gtaaggatct ccaggcatca aataaaacga aaggctcagt cgaaagactg
     6301 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagag accctgacag
     6361 ctagctcagt cctaggtata atgctagcgg gtactaatag cgccgcagtt catgctcttt
     6421 aagagtctct gtactgtgga aaatctgggt tagtttgacg gttggaaaac cgttttgctt
     6481 tctgaccctg gtagctgccc gcttctcatg ctctgacttt tcacgttatg tggaaaaagt
     6541 aacgtaattt cgttagttaa gacttaccgt aaaaagtcag ttctgatgct gctgtcgcaa
     6601 gacaggatag gtgcgctccc agcaaaagga gtatgtcttg aaaaagacta gccgttctag
     6661 taacggtgcg gattaccgca gtggtggcta ctgaatcacc cccttcgtcg ggggaaccct
     6721 ccaaaaggtg ggttgaaagg aagagcgaga tctcgagctc ttcggctcac cttcgggtgg
     6781 gcctttctgc gcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     6841 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     6901 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     6961 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     7021 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     7081 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     7141 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     7201 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     7261 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     7321 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     7381 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     7441 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     7501 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     7561 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     7621 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     7681 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     7741 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     7801 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     7861 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     7921 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     7981 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cg
//
