LOCUS       sequence-354384-        6884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of S. thermophilus type I-E cas8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6884)
  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 6884)
  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_Cas8
FEATURES             Location/Qualifiers
     source          1..6884
                     /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             1828..1836
                     /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    1862..1886
                     /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(1886..1905)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1887..1905)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1989..2008)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2214..2291
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2292..3374
                     /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(2311..2330)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4183..4374
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4368..4390)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4476..4615
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4531..4550
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4630..4647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4801..5389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4881..4900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5500..5519)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5511..6326
                     /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             5511..6326
                     /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(5584..5603)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6418..6873)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6555..6576)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6767..6786
                     /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 ggtggtgttt caattgctgc cgcagccaga
      181 agtcaaacga attataagct gtagctatgt tcttgatgcg accatctttc tgtatgccca
      241 gataatcacg agcaccgccc tgatgtaaaa taacctcggc ctcggctttg acaatttttt
      301 tcaatagtac gcgccattcc tggattttcg aatccttatc atcttcaggc tggatagatg
      361 ccagccactg acggaacggc tggtcaattt taaagtacag cgactccact atttgctgtg
      421 tgtaactgcc attcgaaatg ttgcggattt ctttgatgtc attaatgtaa gttttgtacg
      481 tccggctgat gattttcttc gtctcttcaa ctacttcatt aatccgcggc acccaaccgc
      541 cttcgtccaa atcagtcagc actagatcat taataaaaat agaatcaata acttcatccg
      601 tcggtaaacg tgattgcgga ttcccgtcgt cctgcataga aattgccact atgtttgaca
      661 gacgattacc aataatatca tcgatatacg ttaaccattc gattacaccc ggcttccgat
      721 gctgaagatc agtatccttt atgctgagca gaccgaagga tctccaaaga gactgattta
      781 ataagtgttt tttcggagta tagctgtcgc gattttttcc cgagacatta tatttccata
      841 atgtcatcgg ttccaaaaaa ttatcacggt ggtttatgtc aggtaatttc acaatatcaa
      901 atgagaacgg tttatttaaa ttaaaatccg ggtcgatata gatccctttg gaccacaccg
      961 tatataaact ggctatgttg gtaatatctt tttcatccag ataagatttg ataatatcgc
     1021 tggatttccg ctcccagcat ggacgctgaa tgttttcaac gttgccacat tcattatagg
     1081 gggatatata attcagaagg agagtttcga ataacgtgtt ccccttaata taaatcgcac
     1141 caatatcgaa cagccagccc tttgaagggg tatatttctc gttaccaaag atgactttat
     1201 ccgacacgcc gctatacccc tgaaatgtga ttaaccaacg tgctaattct gccgctgtca
     1261 gggtgttttt attttcttca tctttagggc taaacagggc gattttatta ccggattccg
     1321 aaatgatacg gttaatattt tttcccaata tgctggtgcc cttagctttt gaaattttat
     1381 ctcctgcaat gtcctctttg gtgacctgaa agaatggata tttctgatca aacaggtaaa
     1441 aacgatcacg ccacttttcc agatactgat ttacaatatt cggaaaacgt ttgtttgtcc
     1501 acaatttctc ccaggtttca tacagtgcct cctcgtaatt ttcaatatcc atttcgtcaa
     1561 ccggctcgag ctgattccaa ccctcatcta ttgtcagata ttcataagca ttgccatcga
     1621 tatcaaagcg tgaaaaaact gtatggagca cggccagcag cacacgcaac acagcaaaat
     1681 cttgggtttt tgtgtctccc gctagttctt tgtattgatg agcatttgtg aaaagatcct
     1741 gaagcgaaac atctttcgta ctgccttttt cgtcataaat aaccgaaatc caaggttcat
     1801 ctaaaaggtt aaaccgactc ataaaatacc tccttattat ttgctatctc tatactagag
     1861 gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga
     1921 gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     1981 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
     2041 catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg
     2101 cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact
     2161 actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca
     2221 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     2281 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     2341 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     2401 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     2461 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     2521 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     2581 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     2641 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     2701 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     2761 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     2821 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     2881 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     2941 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     3001 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     3061 ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg
     3121 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     3181 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     3241 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     3301 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     3361 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga
     3421 tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     3481 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     3541 ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc
     3601 ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt
     3661 cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg
     3721 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     3781 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     3841 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     3901 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     3961 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     4021 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     4081 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     4141 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
     4201 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     4261 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     4321 gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     4381 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     4441 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     4501 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     4561 accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     4621 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4681 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4741 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4801 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4861 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4921 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4981 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5041 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5101 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5161 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5221 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     5281 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5341 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5401 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5461 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     5521 ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat
     5581 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt
     5641 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     5701 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     5761 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga
     5821 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     5881 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     5941 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     6001 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     6061 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     6121 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     6181 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     6241 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     6301 atttgatgct cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt
     6361 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     6421 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     6481 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     6541 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     6601 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     6661 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     6721 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     6781 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     6841 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gcca
//
