LOCUS       sequence-354657-        6722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of A. chroococcum type I-E #2 cas8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6722)
  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 6722)
  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: pAc2_Cas8
FEATURES             Location/Qualifiers
     source          1..6722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(111..1193)
                     /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     1155..1174
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1194..1271)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1477..1496
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1580..1599
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1580..1598
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1599..1623
                     /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)."
     CDS             3166..3183
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3236..3254)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3250..3297
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3334..3789
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3421..3440)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3631..3652
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3881..4696)
                     /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(3881..4696)
                     /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     4604..4623
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4688..4707
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4818..5406
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5307..5326
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5560..5577
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5592..5731
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5657..5676)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5817..5839
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5833..6024)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
       61 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
      121 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
      181 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
      241 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
      301 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
      361 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
      421 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
      481 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
      541 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
      601 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
      661 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
      721 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
      781 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
      841 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
      901 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
      961 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     1021 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     1081 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     1141 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     1201 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     1261 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     1321 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     1381 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     1441 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     1501 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     1561 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     1621 tcccctctag aatcgtaagt aaaggcatca aaaggaggta gaatggaaaa tcgctttaat
     1681 ttaatcgacg agccgtggat accagttgcg gatatgggca gagtaagcct gaaagatatt
     1741 ttttcacata gcgaatatag agcgctcggc ggcaacccag tccaaaaaat cgcacttctg
     1801 aaactgctgt tggctatcgc acaggcggcc tgcacgccag aagacgagga agcgtggcgt
     1861 gctctaggct ggcagggttt ggctgctagg tgtcttgatt atctgccgcg ttggcatgaa
     1921 cggttctatc tctatggcga gcgcccgttt ctgcagatgc ctgctatcgc tccggcggca
     1981 ctgcagagtt atggcgcagt aagcccggaa atagctactg gaaacactac tgtcctgacg
     2041 cagtcgcaag tggagagacc tctggatgac gccgacaagg ctctgttact ggtgtgcctt
     2101 atgggtttcg ccctggccgg gaagaaaacc gataattcag tggttttaac cccgggttat
     2161 gccggcaaac gaaacgataa aggtaaaccg agtagtggca aacccggacc agcagtagca
     2221 catatgggac ttctccactc ttgtttccag ggcgaatcac tgatgcaatc cctgtggtta
     2281 aacttactca ctgcgcgtga catagaacgg agtaaattat ttcccgaagg tgtgggtatt
     2341 gcaccgtggg aaaggatgcc ggagggcgaa gggtgccctg ctgcgatggc actccgcggt
     2401 tcattacttg gccgcttagt gcccttgtgc cgcttttgtc tgctgggcga ggaaggcctg
     2461 cattacagcg agggtattgg tcatgccaat tataaagaag ggcttgcaga tccgaccgta
     2521 gcagttgatt tttctggcaa agaaccccgc gccttgtggg tcaatccaga aaaaaaaccg
     2581 tggcgcgaat taacttcact gctaggtttt attcaacagg ataacagagg tggctttgat
     2641 tcgctgcaac tgcggacggg cctgcagaga gtgagggaat cagtggaaca ttttgccatt
     2701 tggtcaggcg gcttgcgcgt ttcatcgaat gcgggggaac agtacgtgag cggttctgat
     2761 gattttgtcg agtcacttgt ttggttacac agtgatatgc ttgggcggtt atggtttgag
     2821 agacttaaag cagagatggc tgatctggac ggcttagcca aaactcttta cggtcgtgtc
     2881 ttaggttatt ttaaagaaca aaagggtgag ggcgaaaaac tggcaggtca gtctacccac
     2941 ttgttttggc agctatgtga gcgcgacttc caggccctgg tagacgcttg tcaaccagat
     3001 gaacaaggtt taggtagaag acagcatctc aggagacgtt ttgcccgtta tcttcagcag
     3061 gcatacgacc gcttttgtcc tcgtgaaaca gcccgtcagc tggacgcatg ggctaaatgc
     3121 cgcccaaaca ccgcgaagta tcttcaacag gaggtttaac tcgagcacca ccaccaccac
     3181 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     3241 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     3301 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     3361 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3421 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3481 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3541 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3601 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3661 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3721 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     3781 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3841 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     3901 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     3961 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     4021 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     4081 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     4141 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     4201 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     4261 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     4321 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     4381 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     4441 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     4501 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     4561 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     4621 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     4681 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     4741 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4801 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4861 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4921 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4981 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5041 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5101 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5161 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5221 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5281 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5341 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5401 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5461 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5521 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5581 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5641 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     5701 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5761 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5821 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     5881 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     5941 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     6001 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     6061 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     6121 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     6181 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     6241 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     6301 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     6361 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     6421 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     6481 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     6541 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     6601 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     6661 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     6721 ag
//
