LOCUS       sequence-447969-        7588 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression and purification of type I-B2 Cascade in 
            CRISPR-associated transposon of Romsetta.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7588)
  AUTHORS   Wang S, Chang L
  TITLE     Biochemical reconstitution of a type I-B CRISPR-associated 
            transposon.
  JOURNAL   Methods Enzymol. 2025;712:55-79. doi: 10.1016/bs.mie.2025.01.042. 
            Epub 2025 Feb 26.
  PUBMED    40121087
REFERENCE   2  (bases 1 to 7588)
  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: "Methods 
            Enzymol."; date: "2025"; volume: "712:55-79. doi"; pages: " 
            10.1016/bs.mie.2025.01.042. Epub 2025 Feb 26"
COMMENT     Sequence Label: pCascade_SPS1(PmcCAST)
FEATURES             Location/Qualifiers
     source          1..7588
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        107..125
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    126..150
                     /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)."
     CDS             858..881
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4059..4077)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4073..4120
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4241..5032)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(5033..5124)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(6185..7267)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7229..7248
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7268..7345)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        7391..7409
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7410..7434
                     /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)."
ORIGIN
        1 atccgaattc gagctcggcg cgcctgcagg tcgacaagct tgcggccgca taatgcttaa
       61 gtcgaacaga aagtaatcgt attgtacacg gccgcataat cgaaattaat acgactcact
      121 ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta agtataagaa
      181 ggagatatac atatgacagc tatattaatt caaagtgagg aatatgtaga cttaacattt
      241 aagctaagag gcgcaccgat tcctcttgac aacggttatc ttacctacgc tgccttatcc
      301 cgtatttgtc ccccactaca tgaactaaag tctattggga ttcatccgat tgctggaata
      361 ccgaccagga acaatttgct tgagctaact gctcaatctc gtctaaaaat tcggatttat
      421 catcagcaaa ttcctttaat ttatccttat ttagcgggtc aagcttttca tataggtcaa
      481 aatttttatc aactagatat ccccgactac aaaccgttaa tttcttcaga aagtgtctat
      541 tccagattag tgataattaa agggttccaa gactctacta actttattga agctgtgcaa
      601 cggcaaatgg ataatttagg aatacaagga aaaattgaac ttcttacccg acaagatgga
      661 acacctcaac gaagacaatt aaccatcaat aaagaaggga aacagtttaa agttagagga
      721 tttggcgtaa aaatcagtga actcaatcct gaagattcct taaccttgca agaacaggga
      781 attggcggga aacgaaagat gatgtgtgga atatttgtcc cagcgactcg cagcaaggaa
      841 gaagaggaaa cctgatgcat catcatcatc atcatcatca tatagtctct acccaaccca
      901 aaatttccct atctctccat gctgcggata cgacaataat gcaccgcgtc ggaatgaccg
      961 gactttacat gactttaaag cgattggaaa agcaatatcc cttatctcgt caacggggag
     1021 gacatatatc atggtttttg actgctgata ctattgaatt attctgggaa ggaagtgatt
     1081 tcatcgcctt atcttggtta attaatgagt cttttcaatt agatgataca ggtttaattc
     1141 atttagtagg attagacaat gatcgaatag atttaaggca gaaaatccat attcatgagg
     1201 gaatatgcgg tgtttttcta cgtcttaata agttttatca agcaggagag ataattaaca
     1261 ctgaactaag gtttgaagaa aaacaagtcg agtatcaata taaatcccta acttggtacg
     1321 cacatcaaac ctttgcagag aagttatgcg aggcagatac ccaacaactc agacatgatt
     1381 acattcaaat aaccagttgg ttgtatttag ggggaattgt tcgccatgct cggacgcaga
     1441 atactacaaa actagaagag aaacctgaat acgctttagc attattattt gtaccagtag
     1501 tttgtcatta ttgcttactt catataccat cagaagactt aaaagaaagg aaaccccatc
     1561 gctacttggt ggtgattcca gaaataaagg attttgaaga cgcttctcaa aggagatggc
     1621 gattacagca attagaaact aaacagtttc atgttagtag ccttggggaa gcaggattac
     1681 tttattacag cttagacgat attcagcctg aagtggccta ctatcaagct tgtcaagttt
     1741 ggttatatga aaaaacaaat aaagcttctc gtcaacggac attgatgagt atagaagaaa
     1801 ttaaaattga taagaatatt ttaatcactt atcaacaggt gcaaaaatac tttaaaacta
     1861 attatcagat aattaaatat aagcaaattt ttattaaggt caatccgatt cgctccctga
     1921 ttgctgataa tttagtgaaa ggaatccatt ggtggtctaa tttttgggaa aaattggtga
     1981 tagaggattc aaaagaatat ttatttaacc agttattttc caatcgagaa ggattcatca
     2041 taatggcaga gaatagtgaa gaagacaagc agtatcttat ttttattaag gtatttcagc
     2101 aagctatgaa gggcaacttt gctaaaattt atgccaaaac agaggaaggt aaagatcctc
     2161 caattaagaa gaaagttgaa cgattaaggg cagagctaaa ctattgttat gatgaattgt
     2221 cgtttaagga atatttgtct gactttttag taagaggggg attaaataag tattttaatg
     2281 agcatcagga agagattgca ctactaatta aaaaatcacc ttggcaagaa ataagaattt
     2341 ggtcattgtt agcgatcgcc agttataaac ccaaagataa actcactaat agggatgata
     2401 gttcattgtc aaacaatcaa aaactagaag aagtgaatga tgactcagaa gaagaatgac
     2461 agtaatatcc caaattatta tctttacgga acagtcctca ctcgttatgg tttagcttcc
     2521 ttaaatcatg acattagacg aggaaataag actattctgc aaaaaggcta ttggaataat
     2581 ggtaaaattc attcttttgt aggttcaagt gcaattcgtt gggcgttacg tttttatctt
     2641 caaaagcaag gttatttagt taatcgagtc tgggatgagg aagaacatat taatcggtta
     2701 acaagtgaag atttcgatcc agaaaaattt tatgatgatg atatttttgg gtttgcttta
     2761 ttagagtctg ctgaaacaga ggaggacact tcaacaacaa agagaaaaaa gaaacaaaca
     2821 aagaccagca ctcctaatca gcgaatgggt gctttaggga tgaatatggc tgtttcgcta
     2881 acgccttatg atggtgcagt taagttaggg gcaaaaagtg gcagagagaa ggatagtaca
     2941 tctctccatt ttacagaata tcatgcaact cgatatcaat attattttgg aattgatgcc
     3001 actcatctca aagatttttc gcggatttta cctatgatag atgggattat gaatctgccc
     3061 aaggtaggag ggagtagtaa tatttttaat tatcctttct gtcccgatag tttggtattt
     3121 caatggacaa atcattttgc ttcatatatt tcctattgct ttgaatattg cgatcctaag
     3181 agtaaggaag caaaactttc acaagagttt atagatgaag tggaatgcgg acagattgac
     3241 cctagtaaat tgtggattgg gggaacaatt gttaaggatt tacagcaatt ggataatttt
     3301 gaaagttccc ctttgaacaa ggcgcatatt tatcgtaatc gaaatgaaat gattgaagct
     3361 ttgaaaacag tcattaaaag agatttgggc ttggaagaat caaaatgatt gcaccattaa
     3421 ttctctattt ggatgttcca tttacgactt ttcgggagtc ccatgcaagg gaaatgggaa
     3481 aaacctatcc tgttcctcct ccagcgacag tgtatgggat gttgttgtct ttggtaggag
     3541 aaacgaatgt ctatcgccac tgtggggtag aattggccat tgcaatgtta tctagcccca
     3601 aaaagtcgcg aattttgcgt caaatgagac ggtttaagaa tgctgatttt agccatccag
     3661 agaatgttat tccctgttat caagaaattt tgtctaatct gaagtgtttg atttgggttc
     3721 gttctgacga ggaaaagata caaccaagtt taagggagag gatacagtta gcgtttgacc
     3781 atcctgagct agtaagacgg tttggttgtt tatttttggg agaaagtgac cagttgatta
     3841 aaacgatcaa acttgcaagg gaagattatc tagagggagt gagacagtgg gcgattagag
     3901 ataatcgagg caggttaact ttaccctatt gggttgacca tgtggggtca agaaataccc
     3961 gatttttgag gtatcggatt gaggaaatgg atagattatc acctcctgat ttagcctgga
     4021 caatggttca atctccgatt tgacctaggc tgctgccacc gctgagcaat aactagcata
     4081 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga
     4141 gaagcacacg gtcacactgc ttccggtagt gccctgaacc gacgaccggg tcatcgtggc
     4201 cggatcttgc ggcccctcgg cttgaacgaa ttgttagaca ttatttgccg actaccttgg
     4261 tgatctcgcc tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc
     4321 gatcttcttc ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg
     4381 ccggcaggcg ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta
     4441 ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt
     4501 cgggcggcga gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag
     4561 gaaccggatc aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg
     4621 cttttgtcag caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa
     4681 gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg
     4741 gaatgatgtc gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc
     4801 caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa
     4861 gccttacggt caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca
     4921 ctgcggagcc gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc
     4981 caactacctc tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc
     5041 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5101 aatgtattta gaaaaataaa caaatagcta gctcactcgg tcgctacgct ccgggcgtga
     5161 gactgcggcg ggcgctgcgg acacatacaa agttacccac agattccgtg gataagcagg
     5221 ggactaacat gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc ataggctccg
     5281 ccctcctgcc agagttcaca taaacagacg cttttccggt gcatctgtgg gagccgtgag
     5341 gctcaaccat gaatctgaca gtacgggcga aacccgacag gacttaaaga tccccaccgt
     5401 ttccggcggg tcgctccctc ttgcgctctc ctgttccgac cctgccgttt accggatacc
     5461 tgttccgcct ttctccctta cgggaagtgt ggcgctttct catagctcac acactggtat
     5521 ctcggctcgg tgtaggtcgt tcgctccaag ctgggctgta agcaagaact ccccgttcag
     5581 cccgactgct gcgccttatc cggtaactgt tcacttgagt ccaacccgga aaagcacggt
     5641 aaaacgccac tggcagcagc cattggtaac tgggagttcg cagaggattt gtttagctaa
     5701 acacgcggtt gctcttgaag tgtgcgccaa agtccggcta cactggaagg acagatttgg
     5761 ttgctgtgct ctgcgaaagc cagttaccac ggttaagcag ttccccaact gacttaacct
     5821 tcgatcaaac cacctcccca ggtggttttt tcgaaggtta agtcagttgg ggaactgctt
     5881 aaccttcgat caaaccacct ccccaggtgg ttttttcgtt tacagggcaa aagattacgc
     5941 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tactgaaccg ctctagattt
     6001 cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt
     6061 aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     6121 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     6181 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6241 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     6301 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     6361 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     6421 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     6481 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     6541 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     6601 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     6661 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     6721 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     6781 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     6841 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     6901 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     6961 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     7021 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     7081 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     7141 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     7201 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     7261 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     7321 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     7381 attaggaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta
     7441 gaaataattt tgtttaactt taataaggag atataccatg gtgagtcgtg gctttatgcc
     7501 gttaggcgtt gctcaagaga agtcatttaa taaggccact gttaaacgta ggtgagtcgt
     7561 ggctttatgc cgttaggcgt tgctcaag
//
