LOCUS       sequence-354960-        5850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of S. thermophilus type I-E cas6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5850)
  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 5850)
  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_Cas6
FEATURES             Location/Qualifiers
     source          1..5850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(154..176)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    262..401
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     317..336
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(416..433)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(587..1175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(667..686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1286..1305)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1297..2112
                     /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             1297..2112
                     /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(1370..1389)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2204..2659)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2341..2362)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2553..2572
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2696..2743)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2739..2757
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2810..2827)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3471..3479
                     /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    3498..3522
                     /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(3522..3541)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3523..3541)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3625..3644)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3850..3927
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3928..5010
                     /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(3947..3966)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             join(5819..5850,1..160)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
       61 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
      121 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
      181 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
      241 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
      301 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg
      361 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
      421 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
      481 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
      541 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
      601 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
      661 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
      721 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
      781 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
      841 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
      901 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
      961 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     1021 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1081 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     1141 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     1201 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa
     1261 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     1321 tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     1381 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     1441 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     1501 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     1561 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag
     1621 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     1681 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     1741 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     1801 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     1861 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     1921 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     1981 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     2041 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     2101 gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2161 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     2221 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     2281 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     2341 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     2401 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     2461 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     2521 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     2581 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     2641 gcgccgctac agggcgcgtc ccattcgcca atccggatat agttcctcct ttcagcaaaa
     2701 aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc tcagcggtgg
     2761 cagcagccaa ctcagcttcc tttcgggctt tgttagcagc cggatctcag tggtggtggt
     2821 ggtggtgttc ttcaagtggc acgaccgtca tcaagccgaa tccataagct ttttttttac
     2881 ccacgccctc cgtcaacatt tttttaaaga gcgtgtcgtc cgaaaccgta agggttcctt
     2941 catacacgac cttgctcagg cgtatcggtt tttctggctt ttgaaagact tcataacctc
     3001 gttccacaat gctgaaactt tctccaacta atgagaaccc atttttttca gattgtttta
     3061 ccagaaagtc catctggtgt ttgattgtca catgcggttt aactaccccg cgcttagatt
     3121 tatcaggaga ggtgatacta attaccgggt taagcacgat gcgaaaattc atacggaaac
     3181 cggttttgat agaatcgaga aagtgctgat aatctttagt ttgtgcacta ccaaccacgc
     3241 cgtatttctc gagtttctgc aggttcggtt tttctttgct cacaatgatc aaataatcct
     3301 tgccctggat acggtcaatg cgccagagct ttcgcgtgcg aatgctttgt tcgagctctg
     3361 acggaaaaga ctcttcgacc caagcgtggt atgcaccaac atgtgtcaga tcccgcacct
     3421 ttcggcgatt ataccgatca atttccacgc gagatatata catctaaaaa acctccttac
     3481 cttgtttacc ctagagggga attgttatcc gctcacaatt cccctatagt gagtcgtatt
     3541 aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
     3601 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
     3661 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
     3721 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
     3781 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
     3841 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     3901 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     3961 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     4021 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     4081 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     4141 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     4201 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     4261 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     4321 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     4381 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     4441 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     4501 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     4561 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     4621 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     4681 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     4741 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     4801 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     4861 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     4921 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4981 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     5041 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     5101 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     5161 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
     5221 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     5281 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     5341 catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     5401 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     5461 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     5521 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     5581 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     5641 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     5701 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     5761 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     5821 gaccaaacag gaaaaaaccg cccttaacat
//
