LOCUS       sequence-342987-        5811 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses E. coli type I-E Cas6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5811)
  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 5811)
  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: pEcCas6
FEATURES             Location/Qualifiers
     source          1..5811
                     /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             760..768
                     /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    789..813
                     /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(813..832)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(814..832)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(916..935)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1141..1218
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1219..2301
                     /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(1238..1257)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3110..3301
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3295..3317)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3403..3542
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3458..3477
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3557..3574)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3728..4316)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3808..3827)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4427..4446)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4438..5253
                     /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             4438..5253
                     /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(4511..4530)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5345..5800)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5482..5503)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5694..5713
                     /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 ggtggtgcag gggagcaaga ctcaataaac
      181 cacaacccat ggacttcgca gggccaatac cctgttgaac caagtcgata agagccggtg
      241 catcatttat ggtcagaaca ccctcgaaac agacggtttg aatctttccg ctttttccat
      301 caccagaaaa atactggggc cgttcgctta tcggatggac gtcttctacg cgagcagcat
      361 tgcccaattt tctctgaagc cacgcaattt gttcagcttc cttaattagg ggtaccctac
      421 aacgtttgat gttgcctttg gaatccaggc gtttctgatt atccaggatt gtcttaatag
      481 gattggcgcg caacctgaag tataacggaa cgccaacttg taactgaaac tcgacttgct
      541 ttgttttgat cactgttgct accgctgtac tcacgggcat ctgggccgat tgtagcagga
      601 cgtgacagcc ttccggcgta ttacgttttt cgacatggaa tagaaagtct cttgcagcat
      661 cagggcggtt cgggaacaga tgccataagc cctgatgcaa ctggtataaa tcgcggctcc
      721 aagcgcgtgc aatgatcacc ttcgataaat acataaatta cctccttact tcttctaacc
      781 cctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
      841 ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
      901 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
      961 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     1021 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
     1081 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg
     1141 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     1201 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     1261 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     1321 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     1381 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     1441 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     1501 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     1561 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     1621 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     1681 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     1741 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     1801 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     1861 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     1921 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     1981 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     2041 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     2101 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     2161 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     2221 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     2281 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     2341 accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc
     2401 gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg
     2461 acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac
     2521 gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt
     2581 cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc
     2641 cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg
     2701 ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg
     2761 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
     2821 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     2881 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
     2941 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
     3001 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
     3061 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
     3121 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
     3181 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
     3241 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     3301 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     3361 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     3421 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     3481 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     3541 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3601 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3661 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3721 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3781 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3841 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3901 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3961 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4021 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4081 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4141 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4201 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4261 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4321 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4381 ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     4441 agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct
     4501 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     4561 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     4621 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     4681 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     4741 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     4801 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     4861 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     4921 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     4981 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     5041 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     5101 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     5161 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     5221 cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt
     5281 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5341 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5401 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5461 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5521 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5581 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5641 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5701 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5761 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc a
//
