LOCUS       sequence-354647-        6284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of S. thermophilus type I-E cas7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6284)
  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 6284)
  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_Cas7
FEATURES             Location/Qualifiers
     source          1..6284
                     /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"
     protein_bind    1262..1286
                     /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(1286..1305)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1287..1305)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1389..1408)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1614..1691
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1692..2774
                     /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(1711..1730)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3583..3774
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3768..3790)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3876..4015
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3931..3950
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4030..4047)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4201..4789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4281..4300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4900..4919)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4911..5726
                     /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             4911..5726
                     /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(4984..5003)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5818..6273)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5955..5976)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6167..6186
                     /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 ggtggtgaat gtactgtgcg atcatttcgg
      181 aaaaatcctc cagtaacttc gagacagaat ccttttcctc accgatctgc ttcagagaat
      241 cagattcatg tagggtaacg taaaaggtca ttaatggttt atctacgaat ttttccactt
      301 ttataaactc tttagctagt ttgtctatgc ttttagtgac atagccttcg atgcttttta
      361 caggttcttc aaaggcggag accaaattga ccggacggtc ggagcggacg gtaacaatca
      421 aagtttgcgg taaggtctga tttgcaaatg tgttaacctt cccagttgga aggctcttag
      481 cgaaggcttc gataaacagc gacagactgt tttccaaact ctccttattc cccagctgtt
      541 tcgatagctc atgaatcgcc acattggcat aacggtaaag tgtagaagag ttaaactcaa
      601 cggtgcccag catgccggcg cccgcattat cttcaggggc taagtcgtct accgcagtga
      661 agaaatcgaa ttccgtctgc actggatgtg tagaaatagc atgggccact tgactcgacg
      721 agtcctcatt aagtgtcgga tcttcagcga ccattcggcc aaaaagggca atgtcgatag
      781 ctggatcttc attggccaat ttctgtaatt cctttttgtg cgttatgtta tcatacgcag
      841 cctgggcaag ttttttggcc tgagaatccc ccataaagta aagtgcttta acttcgcctt
      901 tttttttgat ccccgcagcg atcagaactt tatcggcttt ttctaatgca tcctctatgc
      961 ttatggagga atcaagttct acgattttat ccgctacata acgcacgatt tctttcgtac
     1021 gtattccgac attactatgg tcgccgtggg tattaaaata ttcgcgcatg gcttttttcc
     1081 acgcctgtga cgatacacgg cttcgacgaa caccaccata ctgggcggtt ttcggactac
     1141 cggtatcatc ccgattcata tttgacggtg gtaaggactg aatggcgtga atgtccagaa
     1201 aaagcctctg ctcggtggtc ataataacct tccttaattt gtagtgtgta atgtctagag
     1261 gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga
     1321 gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     1381 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
     1441 catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg
     1501 cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact
     1561 actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca
     1621 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     1681 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     1741 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     1801 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     1861 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     1921 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     1981 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     2041 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     2101 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     2161 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     2221 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     2281 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     2341 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     2401 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     2461 ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg
     2521 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     2581 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     2641 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     2701 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     2761 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga
     2821 tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     2881 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     2941 ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc
     3001 ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt
     3061 cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg
     3121 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     3181 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     3241 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     3301 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     3361 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     3421 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     3481 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     3541 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
     3601 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     3661 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     3721 gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     3781 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3841 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     3901 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     3961 accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     4021 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4081 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4141 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4201 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4261 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4321 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4381 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4441 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4501 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4561 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4621 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     4681 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4741 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4801 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4861 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     4921 ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat
     4981 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt
     5041 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     5101 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     5161 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga
     5221 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     5281 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     5341 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     5401 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     5461 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     5521 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     5581 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     5641 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     5701 atttgatgct cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt
     5761 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     5821 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5881 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5941 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     6001 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     6061 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     6121 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     6181 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     6241 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gcca
//
