LOCUS       sequence-343135-        6715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses E. coli type I-E Cas8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6715)
  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 6715)
  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: pEcCas8
FEATURES             Location/Qualifiers
     source          1..6715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(25..72)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     68..86
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(139..156)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1669..1677
                     /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    1692..1716
                     /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(1716..1735)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1717..1735)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1819..1838)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2044..2121
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2122..3204
                     /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(2141..2160)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4013..4204
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4198..4220)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4306..4445
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4361..4380
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4460..4477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4631..5219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4711..4730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5330..5349)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5341..6156
                     /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             5341..6156
                     /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(5414..5433)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6248..6703)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6385..6406)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6597..6616
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tccggatata gttcctcctt tcagcaaaaa acccctcaag acccgtttag aggccccaag
       61 gggttatgct agttattgct cagcggtggc agcagccaac tcagcttcct ttcgggcttt
      121 gttagcagcc ggatctcagt ggtggtggtg gtggtgacca ttactaggcc cgccctgcgg
      181 tttgagttca cggaggtgtt tatacaaggt tgcgcgtgcc agtgccaaag tggagataag
      241 ttttggatga tgggcatatg gggccacgga ctggttaaac aacatttcgc agagctgatg
      301 caacttatca cgcaaatcgg ctatcacttc gtcagcttga ctgaagttga cgttcgctaa
      361 aacatccgga atcaggagtt ctgactgtct ataaaagtgg cgctcggctg tttcatgaac
      421 gctcactcca gcacctttaa aatctttgtt tttgaaccct tcggcaaagg tgtacagcgc
      481 tttacgaagt gcagttttat agcccaatcc gactgttacg atttcattaa tcacattacc
      541 atattgctgc caaccttgat taaacatcag cacatcgtgg cgacgttcaa ggatggatgc
      601 ttgattattc ctgtagccac ccataatcag ctccagggga ctctgcggtg caatattccg
      661 gaactgattc actactgctg ccacgcggtt gccgttctcg ttctgaataa ttttatctac
      721 aactacgcgg ctaatctggg tccaagaagg tgcagaagtc gtaaatgcca ggaacttttc
      781 ctctacttct ccttttttta cggtgaccag acacggcgaa tgtggatgcg gccacaagcc
      841 attaacagtg aaggtgaatt tttctttaag aaatccagtg taccgtaaat tagactcctg
      901 gccgcagcag ctgcatttgc ctataccaat agggtcacat aactcgatat gtgcaggctg
      961 ccaaaataga ccccgtacaa atccaatgct tgaggctggg attgattcgt ttgacttaat
     1021 cggtttaatc catgtgggtt ggttctcggt atgagattca ttagggaact gtttctgcag
     1081 gcggggcagt gtcagtacat taaggagcac tgtggacctg agatcaatcc ctctgacaaa
     1141 tgttgtaacc ggtgtaccac ctcgcagccc tgatttgaag ccacctccaa agcccggcgc
     1201 ctgattagct tgattgaata acgcaatcgc cgtacagcca ccacacagcg cctctccttg
     1261 acccggctgg ttcacaaacg cgcagttcgt tgcgcctgac acgcctgcta atagtttttc
     1321 cattggggtg acgtcgtttg ctttcacccc tttcgtctgc ataaaggggt gctcggcatg
     1381 atttaagtaa aacatgtcta tccaaggcgc aattaactgt tgaaactcat cctctgttaa
     1441 gggattcatg atacggtgac ggaactcgac atcatctttg gccggtgcaa taatctgtcc
     1501 gatgcaaact aatagggcca gtgccgcgag ctccatatca tcgcgtggca gtgacaggcg
     1561 ccactgatct cggctgcagt ataagctctg caggttaata atctgtactt ttccgccatt
     1621 ccgtgggcgc accggaatcc aattatcaat taacagattc ataaaaaaac ctccttaacg
     1681 gctactagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
     1741 gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     1801 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     1861 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     1921 gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc
     1981 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc
     2041 ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     2101 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     2161 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     2221 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     2281 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     2341 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     2401 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     2461 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     2521 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     2581 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     2641 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     2701 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     2761 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     2821 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     2881 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     2941 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     3001 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     3061 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     3121 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3181 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta
     3241 ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg
     3301 ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt
     3361 aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc
     3421 gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt
     3481 cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc
     3541 ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg
     3601 ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg
     3661 ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta
     3721 aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg
     3781 atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc
     3841 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg
     3901 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt
     3961 catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa
     4021 acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct
     4081 ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga
     4141 ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     4201 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     4261 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     4321 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     4381 ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     4441 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4501 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4561 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4621 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4681 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4741 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4801 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4861 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4921 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4981 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5041 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     5101 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5161 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5221 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5281 ggattttggt catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     5341 atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat
     5401 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc
     5461 tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc
     5521 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct
     5581 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg
     5641 atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt
     5701 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct
     5761 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg
     5821 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa
     5881 gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca
     5941 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc
     6001 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     6061 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     6121 ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatga gcggatacat
     6181 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6241 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     6301 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     6361 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     6421 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     6481 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     6541 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     6601 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     6661 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccaa
//
