LOCUS       sequence-263304-        8696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AcCAST and a crRNA targeting PSP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8696)
  AUTHORS   Strecker J, Ladha A, Gardner Z, Schmid-Burgk JL, Makarova KS, Koonin
            EV, Zhang F
  TITLE     RNA-guided DNA insertion with CRISPR-associated transposases.
  JOURNAL   Science. 2019 Jul 5;365(6448):48-53. doi: 10.1126/science.aax9181. 
            Epub 2019 Jun 6.
  PUBMED    31171706
REFERENCE   2  (bases 1 to 8696)
  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"; doi: 
            "10.1126/science.aax9181"; journalName: "Science"; date: "2019-07-5-
            5"; volume: "365"; issue: "6448"; pages: "48-53"
COMMENT     Sequence Label: pHelper_AcCAST
FEATURES             Location/Qualifiers
     source          1..8696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    95..116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(131..136,137..154,155..161)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    169..185
                     /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)."
     primer_bind     174..196
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     193..209
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     193..209
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        join(260..265,266..283,284..290)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             291..302
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        join(3650..3655,3656..3673,3674..3680)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             3681..3692
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        6114..6148
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     primer_bind     complement(6289..6306)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6289..6305)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6298..6320)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6514..6533)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6633..6655
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        6779..6883
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6884..6952,6953..7744)
                     /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(7102..7121)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7915..8503
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8404..8423
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8657..8674
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
       61 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      121 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      181 aacaatttca cacaggaaac agctatgacc atgattacgc caagctccaa aacgatctca
      241 agaagatcat cttattaatt ttacacttta tgcttccggc tcgtatgttg aaagaggaga
      301 aaggatctat ggcagacgaa gaatttgaat ttactgaagg aacgacgcaa gttccagatg
      361 ctattttgct tgacaagagt aattttgtgg tagatccatc ccaaattatt ctggcaacgt
      421 cggatagaca taaactgaca tttaatctaa tccagtggct tgctgaatct cccaaccgca
      481 ctattaagtc tcagagaaaa caggcagttg caaataccct tgatgtttct actcgccagg
      541 tggaacgtct tctcaagcaa tacgatgaag acaagttaag agagacagca ggaatagaac
      601 gagccgataa gggaaaatat cgagttagcg aatattggca aaacttcatc acaacaatct
      661 atgaaaagag tctgaaagaa aaacatccaa tatcaccagc atccatagtt cgtgaagtga
      721 agcgacacgc aattgtggat cttgaactta agctaggaga atatcctcat caagccactg
      781 tttatagaat tttagatcct ttaatcgagc aacagaaacg gaaaacaaga gttagaaatc
      841 cgggttcggg atcttggatg acagtagtaa cacgagatgg agagttactt agggctgact
      901 ttagtaacca aattattcag tgtgaccata ctaaattgga tgttcgcata gttgataatc
      961 atggcaattt actgtctgat cgtccttggc taactactat tgtggatact ttttcaagct
     1021 gtgttgttgg ttttcgctta tggattaaac aacccggttc tacagaggtg gctttagctt
     1081 taagacacgc tattttacct aaaaactacc ctgaagatta tcaacttaat aagtcttggg
     1141 atgtatgtgg acacccctat caatattttt ttactgatgg tggtaaagat tttcgctcaa
     1201 aacatctcaa agctattggt aagaaattag gatttcagtg tgaattacgc gatcgcccac
     1261 cggaaggtgg tattgtggaa cggattttca aaactattaa tactcaagtt ctcaaagagt
     1321 tacctggtta tacaggggca aatgttcagg aacgcccaga aaatgcagag aaagaagcct
     1381 gtttaactat tcaggatttg gataagattc tcgctagttt cttttgtgat atctataatc
     1441 acgagcctta tcctaaagag cctcgtgata cgagatttga acgctggttt aagggtatgg
     1501 gaggaaaact acctgaacct ttggatgagc gagaattaga tatttgtttg atgaaagaag
     1561 cccaacgagt tgttcaagct catggatcta ttcaatttga aaacctgatt tatcggggag
     1621 aatttctcaa agcacataaa ggtgaatatg taacgctgag atatgatcca gatcatatcc
     1681 tgagtttata tatctacagt ggtgaaactg atgataatgc aggagaattt ttgggttatg
     1741 ctcatgccgt taatatggat acccatgatt taagtataga agaattaaaa gccctgaata
     1801 aagagagaag taatgctcgt aaggagcatt ttaactatga tgctttatta gcattgggta
     1861 aacgtaaaga acttgtagag gaacggaaag aggataaaaa ggcaaaaaga aactcagaac
     1921 aaaagcgtct ccgttctgca tccaagaaaa attccaatgt tattgaacta cgcaaaagta
     1981 ggacttccaa atctttgaag aaacaagaaa atcaggaagt tttaccagag agaatttcca
     2041 gggaagaaat caagcttgag aagatagaac agcaaccaca ggaaaatcta tcagcttcac
     2101 ctaacactca agaagaagag agacataagt tagttttctc taaccgtcaa aaaaatttga
     2161 acaagatttg gtaaattatg gcgcaacctc aacttgcaac tcaatctatt gttgaagtcc
     2221 tagccccaag gttagacatc aaagctcaaa ttgctaaaac tattgatatt gaagagattt
     2281 ttagagcttg ttttatcact actgatcggg cttcggaatg cttcagatgg ttagatgaat
     2341 tgcgtattct caaacaatgt ggtcgaatca ttggaccaag aaatgtggga aaaagcagag
     2401 ccgcgcttca ctatcgagat gaggataaaa aacgagtttc ctatgtaaag gcttggtctg
     2461 catcgagttc taagcggcta ttttcacaaa tcctgaagga tattaatcat gctgcaccaa
     2521 caggtaaacg acaggattta cgtccaagat tagcgggtag tctggaacta tttggattgg
     2581 aattggtgat tatagataat gcggaaaatc ttcaaaaaga agcactgcta gacttgaaac
     2641 aactttttga agagtgtaat gttcctattg ttttagctgg aggtaaggag ttagatgatc
     2701 ttttacacga ttgtgatttg ttgactaatt tcccaacact ctatgagttt gaacggttgg
     2761 aatatgatga tttcaaaaaa acattaacta caattgaatt ggatgtttta tctcttccag
     2821 aagcatctaa tttagctgag ggcaatattt ttgagatttt agcagttagt acagaagcac
     2881 gaatgggaat tttaatcaag atactaacta aggctgtttt acattctctc aaaaatggat
     2941 ttcaccgagt tgatgaaagt attttagaaa aaattgctag tcgttatggc acaaaatata
     3001 ttcctctcaa aaacagaaat agggattgat gaagatgatg aaattcgccc aaagttaggc
     3061 tatgttgaac cttatgagga ggagagtatt agtcattatc tagggcgttt gcgacggttt
     3121 aaggctaaca gcctaccgtc aggatactct ttgggaaaaa ttgctggact cggtgcaatg
     3181 atttcacgtt gggagaagct ttatttcaat ccttttccta ctctacaaga gttggaggct
     3241 ttgtcctctg tggtgggagt taatgcagat agattaatag aaatgctccc ctctcaggga
     3301 atgacgatga agcctagacc aattaggtta tgtggggctt gttatgcaga atctccttgt
     3361 catcggattg agtggcagtg taaggataga atgaaatgcg atcgccacaa tttacgttta
     3421 ttaataaaat gtactaattg tgaaactcct ttcccgattc ccgcagattg ggttaaaggt
     3481 caatgtcctc attgttccct gccttttgca aagatggcga aaaggcaaag gcgtgattag
     3541 attgtgggaa taaggccatt attacagagt gctttgcaat tcttacggag agatttgcga
     3601 tcgcacctca gttacaaaca agaaagagat gtgaacacca cggaaattat ttacacttta
     3661 tgcttccggc tcgtatgttg aaagaggaga aaggatctat gagcgttatc acaattcaat
     3721 gtcgcttggt tgctgaagaa gacagcctcc gtcaactatg ggaattgatg agtgaaaaaa
     3781 atacaccatt catcaatgaa attttgctac agataggaaa acacccagaa tttgaaacct
     3841 ggctagaaaa aggtagaata ccggctgaat tactcaaaac actgggtaac tccctgaaaa
     3901 ctcaagaacc ttttactgga caacctggac gtttttacac ctcagcgatt actttagtgg
     3961 attatctgta taaatcctgg tttgctttac agaaacgcag aaagcagcaa atagaaggga
     4021 aacagcgttg gctaaaaatg ctcaaaagtg atcaagaact tgagcaagaa agtcaatcta
     4081 gcttagaagt aatccgtaat aaagccactg aactttttag caaatttacc cctcagtccg
     4141 atagcgaagc gctccgtagg aatcaaaatg acaaacagaa aaaggtaaaa aagactaaaa
     4201 aatccacaaa accgaaaaca tcttcaattt tcaaaatttt tttaagcact tacgaagaag
     4261 cggaagaacc tcttactcgt tgcgctcttg catatctact caaaaataac tgtcaaatta
     4321 gtgaactgga tgaaaaccca gaagaattta ccagaaataa gcgcagaaaa gaaatagaaa
     4381 ttgagcgatt aaaagatcaa ctccaaagtc gcatccccaa aggtagagat ttgacaggag
     4441 aagaatggtt agaaacctta gaaattgcca ccttcaatgt tccgcaaaat gaaaatgaag
     4501 caaaagcatg gcaagcagca cttttaagaa aaactgctaa tgttcccttt cctgtagctt
     4561 atgaatctaa cgaggatatg acatggttaa agaatgataa aaatcgtctc tttgtacggt
     4621 tcaatggctt gggaaaactt acttttgaga tttactgcga taagcgtcat ttgcactact
     4681 tccaacgctt tttagaggat caagaaattc tacgcaatag taaaaggcag cactcaagca
     4741 gtttgtttac tctacgctca ggaagaatag cttggttgcc aggtgaagaa aaaggtgaac
     4801 attggaaagt aaatcaacta aatttttatt gttctttaga tactcgaatg ctgactaccg
     4861 aaggaactca acaggtagtt gaggagaaag ttacagcaat taccgaaatt ttaaataaaa
     4921 caaaacagaa agatgatctc aacgataaac aacaagcttt tattactcgt cagcaatcaa
     4981 cactagctcg aattaataac ccttttcctc gtcccagtaa acctaattat caaggtaaat
     5041 cttctatcct cataggtgtt agttttggac tagaaaaacc agtcacagta gcagtcgtag
     5101 atgttgttaa aaataaagtt atagcttatc gcagtgtcaa acaactactt ggtgaaaact
     5161 ataatcttct gaatcgtcag cgacaacaac agcaacgcct atctcacgaa cgccacaaag
     5221 cccaaaaaca aaatgcaccc aactcttttg gtgaatctga attaggacaa tatgtggata
     5281 gattgttagc agatgcaatt attgcgatcg ctaaaaaata tcaagctggc agtatagttt
     5341 tacccaaact ccgcgatatg cgagagcaaa tcagcagtga aattcaatcc agagcagaaa
     5401 atcaatgccc tggttacaaa gaaggccaac aaaaatacgc caaagaatat cgaataaacg
     5461 ttcatcgctg gagttatgga cgattaatcg agagtatcaa atcccaagca gcacaagctg
     5521 gaattgcaat tgaaactgga aaacagtcaa tcagaggcag tccacaagaa aaagcacgag
     5581 atttagccgt ctttacttac caagaacgtc aagctgcgct aatttagttt actaatccga
     5641 accttgacaa tataatatgg atacaacagc gccgtagttc atgctccttg gagtctctgt
     5701 actatgaaaa atctggctta gtttggcagt tggaagactg tcatgctttc tgagcctggt
     5761 agctgcccgc ttctgatgct gctgtcgcaa gacaggatag gtgcgctccc agcaataagg
     5821 agtaaggctt ttagccatag tcgttattta taacgatgtg gatttccaca gtggtggcta
     5881 ctgaatcacc cccttcgtcg ggggaatcct cccaaatctt ttttttggcg aaccataagc
     5941 ggggtcaaaa accctgggga tctgccaaaa ccttgaatcc cttgtccagt atttatttga
     6001 ctcatttgaa gagtgatgaa tgtcctctat tgagagcaaa aaaacagatt ttttaacagg
     6061 tttgccaaaa tcgcatctgg aaacctgtat tggcaagggt ctagacgggc gcgttgacag
     6121 ctagctcagt cctaggtata atgctagcgt ttcaactacc atcccgacta ggggtgggtt
     6181 gaaaggagaa gtcatttaat aaggccactg ttaaaactcc aaagtttcaa ctaccatccc
     6241 gactaggggt gggttgaaag cggccgacgc gctgggctac gtcaattcac tggccgtcgt
     6301 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     6361 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     6421 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     6481 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6541 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     6601 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     6661 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     6721 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     6781 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     6841 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     6901 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6961 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     7021 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     7081 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     7141 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     7201 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     7261 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     7321 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     7381 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     7441 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     7501 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     7561 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     7621 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     7681 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     7741 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     7801 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     7861 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7921 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7981 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     8041 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     8101 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     8161 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     8221 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     8281 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     8341 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     8401 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     8461 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     8521 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     8581 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     8641 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatac
//
