LOCUS       sequence-354956-        5939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of S. thermophilus type I-E cas5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5939)
  AUTHORS   Wimmer F, Mougiakos I, Englert F, Beisel CL
  TITLE     Rapid cell-free characterization of multi-subunit CRISPR effectors 
            and transposons.
  JOURNAL   Mol Cell. 2022 Feb 16. pii: S1097-2765(22)00102-2. doi: 
            10.1016/j.molcel.2022.01.026.
  PUBMED    35216669
REFERENCE   2  (bases 1 to 5939)
  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: "Mol Cell. 
            2022 Feb 16. pii: S1097-2765(22)00102-2. doi: 
            10.1016/j.molcel.2022.01.026."
COMMENT     Sequence Label: pSth_Cas5
FEATURES             Location/Qualifiers
     source          1..5939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             886..894
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     protein_bind    917..941
                     /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     complement(941..960)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(942..960)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1044..1063)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1269..1346
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1347..2429
                     /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     complement(1366..1385)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3238..3429
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3423..3445)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3531..3670
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3586..3605
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3685..3702)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3856..4444)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3936..3955)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4555..4574)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4566..5381
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4566..5381
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4639..4658)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5473..5928)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5610..5631)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5822..5841
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgatc gcctatggat tcaaatatat
      181 tgtgttctgt atttttattc aaataatgcg ggttctgcac actgattagt tctcggacct
      241 cgtagcggaa cccgaagcga cgaccttttt gagaaaagga gcgtacatag tcttgccgta
      301 attgatatac atctttattg actaactgac tatccaggta cacttctagt gaaatggcat
      361 ctttatcgtg gctttttttc atgaaccaga gtgaagcttg ccatttaaga gatttcaggg
      421 attccagggg gttgacatcg gtttcgccaa ggataaaatc agccgtaatt ggcaacgcac
      481 ggcgacccat gaacggttgg aaatacgggt ttttgagtgc tcctttaata atatcaatga
      541 aactgtcgtc ggtatgggag agcgccacta caaacacagc gtcttctaga taataacgat
      601 ttgtaacata attccgatcg aatttacctt tttcagtata tttctgggcc acatgaaagt
      661 ctcgaagcaa gttcccctgc tgatctacac ggaccgcaaa atcgagctca ttaagttttt
      721 gtatcttctc gtcctcatcc cgtctatacc ctaaacttgc agccaggaga ccgatgactg
      781 cgcttttaga cgggtaaaag tcggtgtgcc tggtttcaaa atgtgagctg gtcccccaac
      841 tttgcagagg acctgcaaac tttaacagga ttgttttcat aaattacctc cttattggac
      901 taatttaggc tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta
      961 atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg
     1021 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc
     1081 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
     1141 cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa
     1201 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg
     1261 agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     1321 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     1381 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     1441 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     1501 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     1561 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     1621 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     1681 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     1741 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     1801 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     1861 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     1921 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     1981 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     2041 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     2101 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     2161 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     2221 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     2281 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     2341 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2401 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     2461 cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc
     2521 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
     2581 ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg
     2641 agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa
     2701 gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc
     2761 atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     2821 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     2881 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     2941 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     3001 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     3061 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     3121 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     3181 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     3241 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     3301 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     3361 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     3421 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3481 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     3541 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     3601 ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     3661 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3721 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3781 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3841 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3901 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3961 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4021 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4081 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4141 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4201 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4261 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     4321 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4381 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4441 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4501 ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     4561 gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta aattccaaca
     4621 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     4681 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     4741 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     4801 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     4861 ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     4921 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     4981 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     5041 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     5101 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     5161 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     5221 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     5281 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     5341 ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt catgagcgga
     5401 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5461 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     5521 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     5581 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     5641 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     5701 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     5761 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     5821 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     5881 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
