LOCUS       sequence-355202-        8777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Rocas6, Rocas8, Rocas7, Rocas5, RoCAST CRISPR repeat, 
            gfp from Rippkaea orientalis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8777)
  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 8777)
  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: pRoCascade_gfp
FEATURES             Location/Qualifiers
     source          1..8777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(284..1366)
                     /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     1328..1347
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1367..1444)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1650..1669
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1753..1772
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1753..1771
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1772..1796
                     /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             5652..5669
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5722..5740)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5736..5783
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5868..5902
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             6098..6109
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             6116..6829
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      6841..6912
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6928..6955
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(7182..7973)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(7974..8065)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(8113..8777,1..74))
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 cccaggtggt tttttcgttt acagggcaaa agattacgcg cagaaaaaaa ggatctcaag
       61 aagatccttt gatcttttct actgaaccgc tctagatttc agtgcaattt atctcttcaa
      121 atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc
      181 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
      241 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
      301 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      361 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
      421 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
      481 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
      541 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
      601 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
      661 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
      721 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
      781 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
      841 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
      901 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
      961 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     1021 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     1081 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     1141 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     1201 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     1261 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     1321 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     1381 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     1441 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     1501 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     1561 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     1621 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     1681 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     1741 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     1801 taggatccat cttagtatat tagttaagta taagaaggag atatacatat gacagataca
     1861 gttattaaca gtgaagaaca tctagatatg atttttaagc tgaggggtgc accgattcct
     1921 cttgataatg gctatgttat ttattctgct ttgtgtcgaa tttgcccgac gcttcatgaa
     1981 ctcaaagacg tggggattca tcccattgct gggattccta ctcagaataa gtttcttgaa
     2041 ctgactccta attcttgtct aaaaattcgc attcatcatt tacaaattcc tataatttat
     2101 ccttacctaa caggtcaaac ttttgaaatt ggacaagaat cttatcaact gaatattccc
     2161 gattatcaag caatacgttc ctcagatagt ctttattctc gattagtcgt tattaaaggt
     2221 tatcaagact cctctagctt tcttgaagca gttcagcgtc aaatggatag tttagcaata
     2281 caagggaatg tagaacttct gactcgacag aatggaacac cgcaacgacg acaattgacc
     2341 attcgtaagc aaggcaatga gtttaaaata agagggtttg gcgttaagat aagcgaactg
     2401 agtccagagg actctttaac cttgcaagaa caaggaatcg ggggaaaacg gaaaatgatg
     2461 tgtgggatat ttgttccggc gactcgtcct aaagatgaga ggaaaacccc atgacggttg
     2521 caactcaaag caaaatttcc ttatctttga ctgcttgtga tactaccatc atgcaccgtg
     2581 caggaatggc cggactttac ctaactttaa agcgattaga agaaaaatat cctgattttc
     2641 gtcagcgtga gggaaattta tcatggtcat taacccctga tactattgat ttattttggg
     2701 aagcaattga ttttagtgcg ttggattggt tgctgaagga atcttttcaa ctggatgata
     2761 atgggttgat ttgtttgaca ggattagatg attattcaga gttgagacaa aaaattgagc
     2821 ttaatgaagc aataaaagca gtttttctcc gtcataataa attttatcaa gcaggagaag
     2881 ctaaaaaaat ctctttcacc gttgaagaac gtgaagtttc ctattcctac aaacctctaa
     2941 tatggtatgc acatcaaacc tttgctaaag aattatgcga cacagatact caacaattaa
     3001 aacaggatta tcttcaaatt accagttggt tgtatttagg gggaattgtc cgtcatgcga
     3061 gagcagacaa aaagactaaa ctagaagaaa agcctgaata tgctttagcc ttgttatttg
     3121 ttcctgttgt ttgtcattat tgtctttttc accttccgtc tgaggatgta aattcaagga
     3181 aacctcatcg ttatcttgtt gtgattccag aaattaataa ttttgaagat gcttctcgaa
     3241 gaagatggcg attacagcaa ttagaacctc aacaacttca tgttactaat atgagcgaag
     3301 cgggactgct ttattatagt ttggatgaaa ttcaagcaaa tgaaaattat agtcaagctt
     3361 gtcaagtttg gttgtatgag aaaatgaata aagcatctta tcaacgagtg ttgacgatgg
     3421 ttgaagagat tgaaattgat aaggaaatga ttactaccta tcaacaagtt aaacaatact
     3481 ttcaacccaa ttatcaacat atttacccta ataatattct gattgaggta aatctggttc
     3541 ggtcactaat tgcagaaaat ttaaggcaaa aacagccttg gtggtttaat ttttggaaga
     3601 aattgattga tgaagattct aaaaactatt tatttaaaca gttattatct aatcaaaaag
     3661 gggttaaatt catgacaaaa aatagtgaac gagatgaaaa atatctcgct tttattgagg
     3721 tatttcagca agcgatgagg gcaaattttg ccaaaatcta tgcaaaaact gagagaggaa
     3781 aaaaccctct ctttcagaag aaagcagaac gattacgggc agagttaaat tgttgctatg
     3841 atgagatatc atttcagcac tatttagctg attttttagc aagaggcgga ttaaataaac
     3901 cattacaagg tcagaaaaga gaggttttat tattgattca acaagtgcct tgggaaaaat
     3961 taagaatttg ggcattatta gggattgcta gttacgaacc taaacccaaa aatttgcaag
     4021 aattagaagg aattaaagat gaatcagaag aagaataaaa taccaaatta ttatctttat
     4081 ggaacagttt tgactcgcta tggcttagcc tctttaagtc acgggatgcg acaaggtaat
     4141 aaatccattt tacaaaaagc ctattgggat ggcaaaattc attctttagt ggggtcaagt
     4201 tctctacggt gggcgttacg gttttatctt caagaagaag gttatcctgt taacagagta
     4261 tgggataaag atgaacatat taatcgatta acggatgata attttaaccc agatcaattt
     4321 tatgatgatg atatttttgg ctttgcgtta ttggaatctg tagaagaaga aacggtagaa
     4381 gataaaacac aaccatcaac tcgaagacga ggaaaacgaa agaaatcagg gacgactgct
     4441 cagagaacag gggcattaag tatgaataat gctatttctt taactcccta tgacggttct
     4501 ttgaagttag gggcaaaaag tggtagggat aaggatagca catcgcttca ttttacggaa
     4561 tatcataata ctcgttatca atattatttt gggcttaatg ctactcatct taaagattgt
     4621 tctcggattt ttccattaat tgatgggatt atggatatgc cgaaagtggg aggaagtgct
     4681 aatattttca gttatccttt ttgtcctgat agtttagttt ttcaatggac aaatcatttt
     4741 gcgtcttata tttcctattg ttttgaatcc gttgatggga aagggagaaa tattaaattg
     4801 actcaagatt ttatcaatga ggtggaatgc ggacagatta atcctgatga gttatggatt
     4861 ggaggggcaa ttgttaatga tttacgagaa ctagaaaatt ttgagaaatc cttactaaaa
     4921 cagatacatc tttatcgaaa tcgtaatgag ttggtagccg atttaaagga agtgattaaa
     4981 caagatttgg ggttaaaaga ctcacaatga atacaccttt agttatctat ctagatgttc
     5041 cttttgtgac gtttagggag tttcatgcaa gggagatggg aaggacttat cctgttcctc
     5101 ctccagcgac ggtatatggg atgttattgt ctttggtggg ggaaacggat gtttatcgtc
     5161 attgtggggt agaattggca attgccatgt tatctgaagc aaaaaaatcg cggattttgc
     5221 gtcaaatgag acggtttaag cagaaggatt ttagtcatgc agagaatgtg aatcctagct
     5281 atcaggagat tttgtcgaat ttgaagtgct taatttgggt tcgttctgag gctgagatga
     5341 tgcaaccaag cttaaaggag aggatagagt ttacttttga gcgtcctgag ttagtaagac
     5401 ggtttgggtg tttattttta ggggaaagtg accagttaat taagacgata cggttagttt
     5461 ctgaggatta tcgggagggg gtcagacgat ggttaataag ggataatgga ggtaggttaa
     5521 cgttacccta ttgggttgac catctggggt caagaaatac ccgttttttg cggtatcggg
     5581 tagaagaaat gaatcagtta tctcctcctg atttcgcatg gacttcaatt caatcatcgg
     5641 tttgactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     5701 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     5761 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccgaaacc tcaggcattt
     5821 gagaagtgat tctgtggata accgtcgaga gcatcgatcc tagcatgttg acagctagct
     5881 cagtcctagg tataatgcta gctgtgattc atggcttcat gccgttaggc gttgctcaag
     5941 gagacccgtg aaagtgaaac gtgatttcat gcgtcatttt gaacattttg taaatcttat
     6001 ttaataatgt gtgcggcaat tcacatttaa tttatgaatg ttttcttaac atcgcggcaa
     6061 ctcaagaaac ggcaggttcg gatcttagct actagagaaa gaggagaaat actagatgcg
     6121 taaaggcgaa gagctgttca ctggtgtcgt ccctattctg gtggaactgg atggtgatgt
     6181 caacggtcat aagttttccg tgcgtggcga gggtgaaggt gacgcaacta atggtaaact
     6241 gacgctgaag ttcatctgta ctactggtaa actgccggtt ccttggccga ctctggtaac
     6301 gacgctgact tatggtgttc agtgctttgc tcgttatccg gaccatatga agcagcatga
     6361 cttcttcaag tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt cctttaagga
     6421 tgacggcacg tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg
     6481 cattgagctg aaaggcattg actttaaaga ggacggcaat atcctgggcc ataagctgga
     6541 atacaatttt aacagccaca atgtttacat caccgccgat aaacaaaaaa atggcattaa
     6601 agcgaatttt aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg ctgatcacta
     6661 ccagcaaaac actccaatcg gtgatggtcc tgttctgctg ccagacaatc actatctgag
     6721 cacgcaaagc gttctgtcta aagatccgaa cgagaaacgc gatcatatgg ttctgctgga
     6781 gttcgtaacc gcagcgggca tcacgcatgg tatggatgaa ctgtacaaat gaccaggcat
     6841 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     6901 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttag
     6961 gtctcttgtg attcatggct tcatgccgtt aggcgttgct caaccagctg cacaaatcct
     7021 caaacctcta gtatccacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag
     7081 caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcatcgtgg
     7141 ccggatcttg cggcccctcg gcttgaacga attgttagac attatttgcc gactaccttg
     7201 gtgatctcgc ctttcacgta gtggacaaat tcttccaact gatctgcgcg cgaggccaag
     7261 cgatcttctt cttgtccaag ataagcctgt ctagcttcaa gtatgacggg ctgatactgg
     7321 gccggcaggc gctccattgc ccagtcggca gcgacatcct tcggcgcgat tttgccggtt
     7381 actgcgctgt accaaatgcg ggacaacgta agcactacat ttcgctcatc gccagcccag
     7441 tcgggcggcg agttccatag cgttaaggtt tcatttagcg cctcaaatag atcctgttca
     7501 ggaaccggat caaagagttc ctccgccgct ggacctacca aggcaacgct atgttctctt
     7561 gcttttgtca gcaagatagc cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca
     7621 agaatgtcat tgcgctgcca ttctccaaat tgcagttcgc gcttagctgg ataacgccac
     7681 ggaatgatgt cgtcgtgcac aacaatggtg acttctacag cgcggagaat ctcgctctct
     7741 ccaggggaag ccgaagtttc caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca
     7801 agccttacgg tcaccgtaac cagcaaatca atatcactgt gtggcttcag gccgccatcc
     7861 actgcggagc cgtacaaatg tacggccagc aacgtcggtt cgagatggcg ctcgatgacg
     7921 ccaactacct ctgatagttg agtcgatact tcggcgatca ccgcttccct catactcttc
     7981 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     8041 gaatgtattt agaaaaataa acaaatagct agctcactcg gtcgctacgc tccgggcgtg
     8101 agactgcggc gggcgctgcg gacacataca aagttaccca cagattccgt ggataagcag
     8161 gggactaaca tgtgaggcaa aacagcaggg ccgcgccggt ggcgtttttc cataggctcc
     8221 gccctcctgc cagagttcac ataaacagac gcttttccgg tgcatctgtg ggagccgtga
     8281 ggctcaacca tgaatctgac agtacgggcg aaacccgaca ggacttaaag atccccaccg
     8341 tttccggcgg gtcgctccct cttgcgctct cctgttccga ccctgccgtt taccggatac
     8401 ctgttccgcc tttctccctt acgggaagtg tggcgctttc tcatagctca cacactggta
     8461 tctcggctcg gtgtaggtcg ttcgctccaa gctgggctgt aagcaagaac tccccgttca
     8521 gcccgactgc tgcgccttat ccggtaactg ttcacttgag tccaacccgg aaaagcacgg
     8581 taaaacgcca ctggcagcag ccattggtaa ctgggagttc gcagaggatt tgtttagcta
     8641 aacacgcggt tgctcttgaa gtgtgcgcca aagtccggct acactggaag gacagatttg
     8701 gttgctgtgc tctgcgaaag ccagttacca cggttaagca gttccccaac tgacttaacc
     8761 ttcgatcaaa ccacctc
//
