LOCUS       sequence-342952-        5889 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses E. coli type I-E Cas5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5889)
  AUTHORS   Wimmer F, Englert F, Beisel CL
  TITLE     A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems 
            with Multi-Protein Effector Complexes.
  JOURNAL   Methods Mol Biol. 2022;2433:391-411. doi: 
            10.1007/978-1-0716-1998-8_24.
  PUBMED    34985758
REFERENCE   2  (bases 1 to 5889)
  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: "Methods Mol
            Biol."; date: "2022"; volume: "2433:391-411. doi"; pages: " 
            10.1007/978-1-0716-1998-8_24"
COMMENT     Sequence Label: pEcCas5
FEATURES             Location/Qualifiers
     source          1..5889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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)."
     RBS             48..56
                     /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)"
     CDS             735..752
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(805..823)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      819..866
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      903..1358
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(990..1009)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1200..1221
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1450..2265)
                     /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             complement(1450..2265)
                     /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     2173..2192
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2257..2276
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2387..2975
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2876..2895
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3129..3146
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3161..3300
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3226..3245)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3386..3408
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3402..3593)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4402..5484)
                     /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     5446..5465
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5485..5562)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5768..5787
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5871..5889,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5871..5889
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agtataaata acatttttaa ggaggttttt
       61 atatgcgtag ctacctgatt ctccgtttag cggggcctat gcaggcgtgg ggccaaccaa
      121 cttttgaagg aacccgcccc accggtcgtt ttccgacgcg atccggactg ctcggattac
      181 ttggtgcctg tctcggcatc caacgtgatg acacctcctc cctgcaggct ctctctgaaa
      241 gtgttcagtt tgcggtaaga tgcgatgaac tgatcctgga tgatagacgt gtgagcgtga
      301 caggtctgcg cgattatcat acagtcttgg gcgctcgtga agattatagg ggtctcaaaa
      361 gtcatgaaac aatacaaaca tggcgggagt atttatgcga cgcatcattc accgtcgcat
      421 tgtggctaac tccccacgcc acaatggtga tctctgaact ggaaaaggct gtgctgaagc
      481 ctcgctatac accatatcta gggcgtcgtt cctgtcctct gacacatccg cttttcctgg
      541 ggacctgtca agcgagtgat ccacagaaag ctctcttaaa ctatgaaccg gtggggggtg
      601 atatctacag tgaagaatct gtcaccggtc accacctgaa atttacagct cgcgacgagc
      661 caatgatcac gctcccgagg cagtttgcat cgcgagagtg gtacgttatt aaggggggaa
      721 tggacgttag ccagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
      781 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
      841 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
      901 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
      961 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1021 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1081 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1141 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1201 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1261 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1321 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     1381 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1441 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     1501 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     1561 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     1621 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     1681 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     1741 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     1801 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     1861 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     1921 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     1981 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     2041 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     2101 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     2161 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     2221 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     2281 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     2341 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2401 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2461 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2521 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2581 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2641 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2701 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2761 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2821 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2881 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2941 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3001 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3061 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3121 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3181 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     3241 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3301 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3361 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3421 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3481 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3541 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3601 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3661 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     3721 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     3781 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     3841 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     3901 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     3961 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4021 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4081 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4141 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4201 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4261 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4321 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4381 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4441 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4501 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4561 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4621 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4681 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     4741 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     4801 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     4861 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     4921 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     4981 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5041 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5101 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5161 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5221 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5281 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5341 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5401 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5461 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5521 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5581 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     5641 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     5701 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     5761 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     5821 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     5881 cactatagg
//
