LOCUS       sequence-354825-        8024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AcCAST. 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 8024)
  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 8024)
  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: pAcCAST-sgRNA_entry (CJT83)
FEATURES             Location/Qualifiers
     source          1..8024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..35)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(189..777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(269..288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(948..1739,1740..1808))
                     /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     1571..1590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1809..1913)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(1940..1967)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        complement(2262..2296)
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      complement(2310..2381)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             4335..4346
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(join(4347..4353,4354..4371,4372..4377))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             7725..7736
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(join(7737..7743,7744..7761,7762..7767))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(7818..7834)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7818..7834)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7831..7853)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7842..7858
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7866..7872,7873..7890,7891..7896))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7911..7932
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcgtattggg ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
       61 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
      121 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
      181 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
      241 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
      301 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
      361 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
      421 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
      481 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
      541 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
      601 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
      661 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
      721 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
      781 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
      841 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
      901 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
      961 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     1021 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     1081 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     1141 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     1201 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     1261 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     1321 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     1381 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     1441 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     1501 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     1561 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     1621 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     1681 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     1741 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     1801 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     1861 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     1921 ccccgaaaag tgccacctgc gcagaaaggc ccacccgaag gtgagccgaa gagctcgaga
     1981 tctcgctctt cctttcaacc catttagggt tcccccgacg aagggggtga ttcagtagcc
     2041 accactgtgg aaatccacat cgttataaat aacgactatg gctaaaagcc ttactcctta
     2101 ttgctgggag cgcacctatc ctgtcttgcg acagcagcat cagaagcggg cagctaccag
     2161 gctcagaaag catgacagtc ttccaactgc caaactaagc cagatttttc atagtacaga
     2221 gactccaagg agcatgaact acggcgctgt tgtatccata tgctagcatt atacctagga
     2281 ctgagctagc tgtcagggtc tctctagtag agagcgttca ccgacaaaca acagataaaa
     2341 cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcctgga gatccttacc
     2401 taaattagcg cagcttgacg ttcttggtaa gtaaagacgg ctaaatctcg tgctttttct
     2461 tgtggactgc ctctgattga ctgttttcca gtttcaattg caattccagc ttgtgctgct
     2521 tgggatttga tactctcgat taatcgtcca taactccagc gatgaacgtt tattcgatat
     2581 tctttggcgt atttttgttg gccttctttg taaccagggc attgattttc tgctctggat
     2641 tgaatttcac tgctgatttg ctctcgcata tcgcggagtt tgggtaaaac tatactgcca
     2701 gcttgatatt ttttagcgat cgcaataatt gcatctgcta acaatctatc cacatattgt
     2761 cctaattcag attcaccaaa agagttgggt gcattttgtt tttgggcttt gtggcgttcg
     2821 tgagataggc gttgctgttg ttgtcgctga cgattcagaa gattatagtt ttcaccaagt
     2881 agttgtttga cactgcgata agctataact ttatttttaa caacatctac gactgctact
     2941 gtgactggtt tttctagtcc aaaactaaca cctatgagga tagaagattt accttgataa
     3001 ttaggtttac tgggacgagg aaaagggtta ttaattcgag ctagtgttga ttgctgacga
     3061 gtaataaaag cttgttgttt atcgttgaga tcatctttct gttttgtttt atttaaaatt
     3121 tcggtaattg ctgtaacttt ctcctcaact acctgttgag ttccttcggt agtcagcatt
     3181 cgagtatcta aagaacaata aaaatttagt tgatttactt tccaatgttc acctttttct
     3241 tcacctggca accaagctat tcttcctgag cgtagagtaa acaaactgct tgagtgctgc
     3301 cttttactat tgcgtagaat ttcttgatcc tctaaaaagc gttggaagta gtgcaaatga
     3361 cgcttatcgc agtaaatctc aaaagtaagt tttcccaagc cattgaaccg tacaaagaga
     3421 cgatttttat cattctttaa ccatgtcata tcctcgttag attcataagc tacaggaaag
     3481 ggaacattag cagtttttct taaaagtgct gcttgccatg cttttgcttc attttcattt
     3541 tgcggaacat tgaaggtggc aatttctaag gtttctaacc attcttctcc tgtcaaatct
     3601 ctacctttgg ggatgcgact ttggagttga tcttttaatc gctcaatttc tatttctttt
     3661 ctgcgcttat ttctggtaaa ttcttctggg ttttcatcca gttcactaat ttgacagtta
     3721 tttttgagta gatatgcaag agcgcaacga gtaagaggtt cttccgcttc ttcgtaagtg
     3781 cttaaaaaaa ttttgaaaat tgaagatgtt ttcggttttg tggatttttt agtctttttt
     3841 acctttttct gtttgtcatt ttgattccta cggagcgctt cgctatcgga ctgaggggta
     3901 aatttgctaa aaagttcagt ggctttatta cggattactt ctaagctaga ttgactttct
     3961 tgctcaagtt cttgatcact tttgagcatt tttagccaac gctgtttccc ttctatttgc
     4021 tgctttctgc gtttctgtaa agcaaaccag gatttataca gataatccac taaagtaatc
     4081 gctgaggtgt aaaaacgtcc aggttgtcca gtaaaaggtt cttgagtttt cagggagtta
     4141 cccagtgttt tgagtaattc agccggtatt ctaccttttt ctagccaggt ttcaaattct
     4201 gggtgttttc ctatctgtag caaaatttca ttgatgaatg gtgtattttt ttcactcatc
     4261 aattcccata gttgacggag gctgtcttct tcagcaacca agcgacattg aattgtgata
     4321 acgctcatag atcctttctc ctctttcaac atacgagccg gaagcataaa gtgtaaataa
     4381 tttccgtggt gttcacatct ctttcttgtt tgtaactgag gtgcgatcgc aaatctctcc
     4441 gtaagaattg caaagcactc tgtaataatg gccttattcc cacaatctaa tcacgccttt
     4501 gccttttcgc catctttgca aaaggcaggg aacaatgagg acattgacct ttaacccaat
     4561 ctgcgggaat cgggaaagga gtttcacaat tagtacattt tattaataaa cgtaaattgt
     4621 ggcgatcgca tttcattcta tccttacact gccactcaat ccgatgacaa ggagattctg
     4681 cataacaagc cccacataac ctaattggtc taggcttcat cgtcattccc tgagagggga
     4741 gcatttctat taatctatct gcattaactc ccaccacaga ggacaaagcc tccaactctt
     4801 gtagagtagg aaaaggattg aaataaagct tctcccaacg tgaaatcatt gcaccgagtc
     4861 cagcaatttt tcccaaagag tatcctgacg gtaggctgtt agccttaaac cgtcgcaaac
     4921 gccctagata atgactaata ctctcctcct cataaggttc aacatagcct aactttgggc
     4981 gaatttcatc atcttcatca atccctattt ctgtttttga gaggaatata ttttgtgcca
     5041 taacgactag caattttttc taaaatactt tcatcaactc ggtgaaatcc atttttgaga
     5101 gaatgtaaaa cagccttagt tagtatcttg attaaaattc ccattcgtgc ttctgtacta
     5161 actgctaaaa tctcaaaaat attgccctca gctaaattag atgcttctgg aagagataaa
     5221 acatccaatt caattgtagt taatgttttt ttgaaatcat catattccaa ccgttcaaac
     5281 tcatagagtg ttgggaaatt agtcaacaaa tcacaatcgt gtaaaagatc atctaactcc
     5341 ttacctccag ctaaaacaat aggaacatta cactcttcaa aaagttgttt caagtctagc
     5401 agtgcttctt tttgaagatt ttccgcatta tctataatca ccaattccaa tccaaatagt
     5461 tccagactac ccgctaatct tggacgtaaa tcctgtcgtt tacctgttgg tgcagcatga
     5521 ttaatatcct tcaggatttg tgaaaatagc cgcttagaac tcgatgcaga ccaagccttt
     5581 acataggaaa ctcgtttttt atcctcatct cgatagtgaa gcgcggctct gctttttccc
     5641 acatttcttg gtccaatgat tcgaccacat tgtttgagaa tacgcaattc atctaaccat
     5701 ctgaagcatt ccgaagcccg atcagtagtg ataaaacaag ctctaaaaat ctcttcaata
     5761 tcaatagttt tagcaatttg agctttgatg tctaaccttg gggctaggac ttcaacaata
     5821 gattgagttg caagttgagg ttgcgccata atttaccaaa tcttgttcaa atttttttga
     5881 cggttagaga aaactaactt atgtctctct tcttcttgag tgttaggtga agctgataga
     5941 ttttcctgtg gttgctgttc tatcttctca agcttgattt cttccctgga aattctctct
     6001 ggtaaaactt cctgattttc ttgtttcttc aaagatttgg aagtcctact tttgcgtagt
     6061 tcaataacat tggaattttt cttggatgca gaacggagac gcttttgttc tgagtttctt
     6121 tttgcctttt tatcctcttt ccgttcctct acaagttctt tacgtttacc caatgctaat
     6181 aaagcatcat agttaaaatg ctccttacga gcattacttc tctctttatt cagggctttt
     6241 aattcttcta tacttaaatc atgggtatcc atattaacgg catgagcata acccaaaaat
     6301 tctcctgcat tatcatcagt ttcaccactg tagatatata aactcaggat atgatctgga
     6361 tcatatctca gcgttacata ttcaccttta tgtgctttga gaaattctcc ccgataaatc
     6421 aggttttcaa attgaataga tccatgagct tgaacaactc gttgggcttc tttcatcaaa
     6481 caaatatcta attctcgctc atccaaaggt tcaggtagtt ttcctcccat acccttaaac
     6541 cagcgttcaa atctcgtatc acgaggctct ttaggataag gctcgtgatt atagatatca
     6601 caaaagaaac tagcgagaat cttatccaaa tcctgaatag ttaaacaggc ttctttctct
     6661 gcattttctg ggcgttcctg aacatttgcc cctgtataac caggtaactc tttgagaact
     6721 tgagtattaa tagttttgaa aatccgttcc acaataccac cttccggtgg gcgatcgcgt
     6781 aattcacact gaaatcctaa tttcttacca atagctttga gatgttttga gcgaaaatct
     6841 ttaccaccat cagtaaaaaa atattgatag gggtgtccac atacatccca agacttatta
     6901 agttgataat cttcagggta gtttttaggt aaaatagcgt gtcttaaagc taaagccacc
     6961 tctgtagaac cgggttgttt aatccataag cgaaaaccaa caacacagct tgaaaaagta
     7021 tccacaatag tagttagcca aggacgatca gacagtaaat tgccatgatt atcaactatg
     7081 cgaacatcca atttagtatg gtcacactga ataatttggt tactaaagtc agccctaagt
     7141 aactctccat ctcgtgttac tactgtcatc caagatcccg aacccggatt tctaactctt
     7201 gttttccgtt tctgttgctc gattaaagga tctaaaattc tataaacagt ggcttgatga
     7261 ggatattctc ctagcttaag ttcaagatcc acaattgcgt gtcgcttcac ttcacgaact
     7321 atggatgctg gtgatattgg atgtttttct ttcagactct tttcatagat tgttgtgatg
     7381 aagttttgcc aatattcgct aactcgatat tttcccttat cggctcgttc tattcctgct
     7441 gtctctctta acttgtcttc atcgtattgc ttgagaagac gttccacctg gcgagtagaa
     7501 acatcaaggg tatttgcaac tgcctgtttt ctctgagact taatagtgcg gttgggagat
     7561 tcagcaagcc actggattag attaaatgtc agtttatgtc tatccgacgt tgccagaata
     7621 atttgggatg gatctaccac aaaattactc ttgtcaagca aaatagcatc tggaacttgc
     7681 gtcgttcctt cagtaaattc aaattcttcg tctgccatag atcctttctc ctctttcaac
     7741 atacgagccg gaagcataaa gtgtaaaatt aataagatga tcttcttgag atcgttttgg
     7801 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     7861 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     7921 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     7981 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gttt
//
