LOCUS       sequence-354401-        5799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of S. thermophilus type I-E cse2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5799)
  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 5799)
  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_Cse2
FEATURES             Location/Qualifiers
     source          1..5799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(182..204)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    290..429
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     345..364
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(444..461)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(615..1203)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(695..714)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1314..1333)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1325..2140
                     /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             1325..2140
                     /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(1398..1417)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2232..2687)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2369..2390)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2581..2600
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2724..2771)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2767..2785
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2838..2855)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    3475..3499
                     /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(3499..3518)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3500..3518)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3602..3621)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3827..3904
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3905..4987
                     /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(3924..3943)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             join(5796..5799,1..188)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
       61 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
      121 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
      181 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
      241 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
      301 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
      361 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
      421 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
      481 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
      541 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      601 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      661 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
      721 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
      781 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
      841 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
      901 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
      961 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1021 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     1081 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1141 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1201 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1261 taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     1321 tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat
     1381 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     1441 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     1501 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     1561 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     1621 tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     1681 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     1741 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     1801 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     1861 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     1921 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     1981 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     2041 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     2101 taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat
     2161 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2221 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2281 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2341 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2401 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2461 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2521 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2581 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2641 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccaat
     2701 ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg
     2761 ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg
     2821 ttagcagccg gatctcagtg gtggtggtgg tggtggttgt cattattctt ttcacctttg
     2881 atctgtttgt aatacgcccg agcccaattc aatcgtacgt tttcctgaga attgtttaag
     2941 aaccagtaaa gatcattcgc aagaccgcta taatcgacct gaataaccgg cgattcaact
     3001 ttcagtaaac ttataaggtg tctaaggtgg tacgtcagtt cctcgaaggt gctgcttgtg
     3061 atcatggtat taaatctgcg gtcaatacta gtggtatcat cttccttgcg cagtttaccc
     3121 agggcggaac cgatattctc atacggttta ctatcatcca gcataacgct cgtcgaaaca
     3181 ccttgctgat gcaatgcata taactgtagg gctgtataaa tagccagttc ttcataacta
     3241 ggttgctgat agctactaag aaattcttca ggcagatttt caaacagtat cggccaaatt
     3301 tcagtcgctt cactcaacgg tttcccgagc gttttccgaa ctttcgccag tgttgctttg
     3361 tcataggtct cttccttctg attgctcagc ttgaatataa tttttttagt gacggtccat
     3421 acggtctgtt tagtatgctg tgacataaaa aatctgctta ttcgagtcta gaggggaatt
     3481 gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg
     3541 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
     3601 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
     3661 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
     3721 tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
     3781 tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg
     3841 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     3901 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     3961 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     4021 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     4081 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     4141 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     4201 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     4261 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     4321 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     4381 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     4441 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     4501 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     4561 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     4621 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     4681 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     4741 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     4801 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     4861 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     4921 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     4981 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac
     5041 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     5101 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     5161 cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt
     5221 cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca
     5281 ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca
     5341 tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     5401 agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     5461 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     5521 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     5581 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     5641 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     5701 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     5761 ccatgaacag aaatccccct tacacggagg catcagtga
//
