LOCUS       sequence-354262-        9189 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  13S-S Candidatus "Scalindua brodae" Twin- Strep SUMO tagged RAMP 
            (codon-optimization).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9189)
  AUTHORS   van Beljouw SPB, Haagsma AC, Rodriguez-Molina A, van den Berg DF, 
            Vink JNA, Brouns SJJ
  TITLE     The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with 
            a caspase-like peptidase.
  JOURNAL   Science. 2021 Sep 17;373(6561):1349-1353. doi: 
            10.1126/science.abk2718. Epub 2021 Aug 26.
  PUBMED    34446442
REFERENCE   2  (bases 1 to 9189)
  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"; doi: 
            "10.1126/science.abk2718"; journalName: "Science"; date: 
            "2021-09-17- 17"; volume: "373"; issue: "6561"; pages: "1349-1353"
COMMENT     Sequence Label: pTU398
FEATURES             Location/Qualifiers
     source          1..9189
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(122..140)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      136..183
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(354..1145)
                     /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(1146..1237)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1285..2023)
                     /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."
     CDS             complement(2232..3314)
                     /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     3276..3295
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3315..3392)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3438..3457
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3438..3456
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3461..3484
                     /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             3537..3559
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3585..3608
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3645..3668
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3687..3980
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3987..4007
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
ORIGIN
        1 attggaagtg gataacggat ccgcgatcgc ggcgcgccac ctggtggccg gccggtacca
       61 cgcgtgcgcg ctgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      121 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      181 ttgctgaaac ctcaggcatt tgagaagcac acggtcacac tgcttccggt agtcaataaa
      241 ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga accgacgacc
      301 gggtcatcgt ggccggatct tgcggcccct cggcttgaac gaattgttag acattatttg
      361 ccgactacct tggtgatctc gcctttcacg taatggacaa attcttccaa ctgatctgcg
      421 cgcgaggcca agcgatcttc ttcttgtcca agataagcct gtctagcttc aagtatgacg
      481 ggctgatact gggccggcag gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg
      541 attttgccgg ttactgcgct gtaccaaatg cgggacaacg taagcactac atttcgctca
      601 tcgccagccc agtcgggcgg cgagttccat agcgttaagg tttcatttag cgcctcaaat
      661 agatcctgtt caggaaccgg atcaaagagt tcctccgccg ctggacctac caaggcaacg
      721 ctatgttctc ttgcttttgt cagcaagata gccagatcaa tgtcgatcgt ggctggctcg
      781 aagatacctg caagaatgtc attgcgctgc cattctccaa attgcagttc gcgcttagct
      841 ggataacgcc acggaatgat gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga
      901 atctcgctct ctccagggga agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc
      961 gttgtttcat caagccttac ggtcaccgta accagcaaat caatatcact gtgcggcttc
     1021 aggccgccat ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg
     1081 cgctcgatga cgccaactac ctctgatagt tgagtcgata cttcggcgat caccgcttcc
     1141 ctcatactct tcctttttca ttattattga agcatttatc agggttattg tctcatgagc
     1201 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag ctagctcact cggtcgctac
     1261 gctccgggcg tgagactgcg gcgggcgctg cggacacata caaagttacc cacagattcc
     1321 gtggataagc aggggactaa catgtgaggc aaaacagcag ggccgcgccg gtggcgtttt
     1381 tccataggct ccgccctcct gccagagttc acataaacag acgcttttcc ggtgcatctg
     1441 tgggagccgt gaggctcaac catgaatctg acagtacggg cgaaacccga caggacttaa
     1501 agatccccac cgtttccggc gggtcgctcc ctcttgcgct ctcctgttcc gaccctgccg
     1561 tttaccggat acctgttccg cctttctccc ttacgggaag tgtggcgctt tctcatagct
     1621 cacacactgg tatctcggct cggtgtaggt cgttcgctcc aagctgggct gtaagcaaga
     1681 actccccgtt cagcccgact gctgcgcctt atccggtaac tgttcacttg agtccaaccc
     1741 ggaaaagcac ggtaaaacgc cactggcagc agccattggt aactgggagt tcgcagagga
     1801 tttgtttagc taaacacgcg gttgctcttg aagtgtgcgc caaagtccgg ctacactgga
     1861 aggacagatt tggttgctgt gctctgcgaa agccagttac cacggttaag cagttcccca
     1921 actgacttaa ccttcgatca aaccacctcc ccaggtggtt ttttcgttta cagggcaaaa
     1981 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta ctgaaccgct
     2041 cttatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat
     2101 aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     2161 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     2221 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     2281 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     2341 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     2401 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     2461 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca
     2521 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     2581 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     2641 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     2701 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     2761 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     2821 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     2881 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     2941 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     3001 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     3061 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     3121 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     3181 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     3241 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     3301 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     3361 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     3421 ctcctgcatt aggaaattaa tacgactcac tatagggaga gaattgtgag cggataacaa
     3481 ttcccctgta gattaattaa gcggccgccc tgcaggactc gagttctaga aataattttg
     3541 tttaacttta agaaggagat atacatatga aatcttcttc cgcgtggtct caccctcagt
     3601 ttgaaaaagg cggcggttct ggcggcggct ccggcggtgg tgcttggtcc cacccgcagt
     3661 ttgaaaaagg ttcttctatg gctagcatgt cggactcaga agtcaatcaa gaagctaagc
     3721 cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg tccgatggat
     3781 cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg atggaagcgt
     3841 tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac ggtattagaa
     3901 ttcaagctga tcagacccct gaagatttgg acatggagga taacgatatt attgaggctc
     3961 acagagaaca gattggtggg atcgaggaaa acctgtactt ccaatccaat gcaatgaaaa
     4021 gcaacgacat gaacattacc gtggaactga ccttttttga accgtatcgt ctggttgaat
     4081 ggtttgattg ggatgcacgt aaaaaaagcc atagcgcaat gcgtggtcag gcatttgcac
     4141 agtggacctg gaaaggtaaa ggtcgtaccg caggtaaaag ctttattacc ggtacactgg
     4201 ttcgtagcgc agttattaaa gcagttgaag aactgctgag cctgaataat ggtaaatggg
     4261 aaggtgttcc gtgttgcaat ggtagctttc agaccgatga aagcaaaggt aaaaaaccga
     4321 gctttctgcg taaacgtcat accctgcagt ggcaggcaaa taacaaaaac atttgcgata
     4381 aagaagaggc ctgtccgttt tgtattctgc tgggtcgttt tgataatgcc ggtaaagtgc
     4441 atgaacgcaa caaagattat gatatccact tcagcaactt cgacctggat cacaaacaag
     4501 aaaaaaatga tctgcgcctg gttgatattg caagcggtcg tattctgaat cgtgttgatt
     4561 ttgataccgg caaagccaaa gattactttc gtacctggga agcagattat gaaacctatg
     4621 gcacctatac cggtcgcatt accctgcgta atgaacatgc aaaaaaactg ctgctggcaa
     4681 gcctgggttt tgttgataaa ctgtgtggtg cactgtgtcg tattgaggtt atcaaaaaaa
     4741 gcgaaagtcc gctgccgagc gataccaaag aacagagcta tacaaaagat gataccgttg
     4801 aagttctgag cgaagatcat aatgatgaac tgcgcaaaca ggccgaagtt attgttgaag
     4861 catttaagca gaacgataaa ctggaaaaaa ttcgcattct ggcagatgca attcgtaccc
     4921 tgcgcctgca tggtgaaggt gtgattgaaa aagatgagct gccggatggt aaagaagaac
     4981 gcgataaagg tcatcatctg tgggatatta aagttcaggg caccgcactg cgtaccaaac
     5041 tgaaagaact gtggcagagc aataaagata ttggctggcg caaatttacc gaaatgctgg
     5101 gtagcaatct gtacctgatc tataagaaag aaaccggtgg tgttagcacc cgttttcgca
     5161 tcctgggtga taccgagtat tatagcaaag cacatgatag cgaaggtagc gacctgttta
     5221 ttccggttac accgcctgaa ggtattgaaa ccaaagaatg gattattgtg ggtcgcctga
     5281 aagcagcaac cccgttttat ttcggtgttc agcagccgag tgatagcatt ccgggtaaag
     5341 agaaaaaatc agaagatagc ctggtcatca atgaacacac cagctttaac atcctgctgg
     5401 ataaagaaaa tcgttatcgt attccgcgta gtgcactgcg tggtgccctg cgtcgcgatc
     5461 tgcgtaccgc atttggtagc ggttgtaatg ttagcttagg tggtcagatt ctgtgcaatt
     5521 gtaaagtgtg tattgaaatg cgtcgcatca cactgaaaga tagcgttagc gatttttcag
     5581 aacctccgga aattcgctat cgcattgcaa aaaatccggg tacagcaacc gtggaagatg
     5641 gtagtctgtt tgatattgaa gttggtccgg aaggcctgac ctttccgttt gttctgcgtt
     5701 atcgtggtca taaatttcca gaacagctga gcagcgttat tcgttattgg gaagaaaatg
     5761 atggcaaaaa tggtatggca tggttaggtg gcctggatag caccggtaaa ggccgttttg
     5821 ccctgaaaga cattaaaatc tttgagtggg atctgaacca gaaaatcaac gaatatatca
     5881 aagaacgcgg tatgcgtggc aaagaaaaag aattactgga aatgggtgaa agcagtctgc
     5941 ctgatggtct gattccgtat aaattctttg aagaacgtga atgcctgttt ccgtacaaag
     6001 aaaacctgaa accgcagtgg tcagaagttc agtataccat tgaagtgggt tcaccgctgc
     6061 tgaccgcaga taccattagc gcactgaccg aaccgggtaa tcgtgatgca attgcctaca
     6121 aaaaacgcgt gtataacgat ggcaataatg ccattgaacc ggaaccgcgt tttgcagtta
     6181 aaagtgaaac ccatcgtggt atttttcgca ccgcagttgg tcgtcgtacc ggtgatctgg
     6241 gcaaagaaga tcacgaagat tgtacctgtg atatgtgcat tatctttggc aatgagcatg
     6301 agagcagcaa aattcgtttt gaagatctgg aactgatcaa cggcaacgaa tttgaaaagc
     6361 tggaaaaaca tattgaccac gtggccattg atcgttttac aggtggcgca ctggacaaag
     6421 caaaatttga tacctatccg ctggcaggta gcccgaaaaa accgctgaaa ctgaagggtc
     6481 gcttttggat taaaaagggt tttagcggtg atcacaagct gctgattacc acagcactga
     6541 gcgatattcg tgatggcctg tatcctctgg gtagtaaagg tggtgttggt tatggttggg
     6601 ttgcaggtat tagcattgat gataatgtgc cggatgactt taaagagatg atcaacaaga
     6661 cagaaatgcc gctgccggaa gaagtggaag aaagcaataa tggtccgatc aataacgatt
     6721 atgttcatcc gggtcatcag agcccgaaac aggatcataa aaacaagaac atctattatc
     6781 cgcattattt tctggacagc ggcagcaaag tgtatcgcga aaaagatatt atcacccacg
     6841 aagaattcac cgaggaactg ctgtcaggca aaattaactg taaacttgaa accctgacac
     6901 cgctgattat tccggatacc agtgatgaaa atggtctgaa acttcagggt aataaaccgg
     6961 gtcataagaa ctacaaattc ttcaacatta atggcgaact gatgattccg ggttcagaac
     7021 tgcgtggcat gctgcgcacc cattttgaag cactgaccaa aagctgtttt gccatttttg
     7081 gtgaagatag caccctgagc tggcgtatga atgcagatga gaaagattac aaaatcgata
     7141 gcaacagcat ccgcaaaatg gaaagccagc gtaatccgaa atatcgcatt ccggacgaac
     7201 tgcagaaaga gctgcgtaat agcggtaatg gtctgtttaa tcgtctgtat accagcgaac
     7261 gtcgtttttg gagtgatgtg agtaacaaat ttgagaacag catcgattac aaacgcgaaa
     7321 ttctgcgttg tgcaggtcgt ccgaaaaact ataaaggcgg tattattcgt cagcgtaaag
     7381 atagtctgat ggccgaagaa ctgaaagttc atcgtctgcc tctgtatgat aactttgata
     7441 ttcctgatag cgcctacaaa gccaacgatc attgtcgtaa aagcgcaacc tgtagcacca
     7501 gccgtggttg tcgtgaacgt tttacctgtg gcattaaagt gcgtgataaa aatcgcgttt
     7561 ttctgaatgc agccaataat aatcgccagt acctgaacaa catcaaaaag tccaatcacg
     7621 atctgtatct gcagtatctg aaaggcgaaa aaaagatccg cttcaacagc aaagttatta
     7681 caggtagcga acgtagcccg attgatgtta ttgcagaact gaatgaacgt ggtcgtcaga
     7741 ccggttttat caaactgagc ggtctgaata acagcaataa aagccagggc aataccggca
     7801 ccacatttaa tagtggttgg gatcgctttg aactgaatat actgctggat gatctggaaa
     7861 cccgtccgag caaaagcgat tatccgcgtc cgcgtctgct gtttaccaaa gatcagtatg
     7921 aatacaacat caccaaacgt tgcgaacgcg tgtttgaaat tgataaaggc aacaaaacag
     7981 gctatccggt ggatgatcag atcaaaaaga actacgaaga tatcctggac agctatgatg
     8041 gcatcaaaga tcaagaagtt gccgaacgct ttgatacatt tacccgtggt agcaagctga
     8101 aagttggcga tctggtttat tttcatatcg acggcgataa caaaattgac agcctgattc
     8161 cggttcgtat tagccgtaaa tgtgcaagca aaaccttagg tggcaaatta gataaagcac
     8221 tgcatccgtg caccggtctg tcagatggtc tgtgtccggg ttgtcacctg tttggcacca
     8281 ccgattataa aggtcgcgtt aaatttggct tcgccaaata tgaaaacggt cctgaatggc
     8341 tgattacgcg tggtaataat ccggaacgta gtctgaccct gggtgtgctg gaatcaccgc
     8401 gtccggcatt ttcaattccg gatgatgaaa gtgaaattcc gggtcgtaaa ttctatctgc
     8461 atcacaatgg ttggcgcatt attcgccaga aacaactgga aattcgtgaa accgttcagc
     8521 cggaacgcaa tgttaccacc gaagtgatgg ataaaggtaa cgtgtttagc tttgatgtgc
     8581 gctttgaaaa tctgcgtgaa tgggaactgg gtctgctgct gcagagtctg gatcctggta
     8641 aaaacattgc acataaactt ggtaaaggca aaccgtatgg ttttggcagc gtgaaaatca
     8701 agattgatag cctgcatacc ttcaagatta acagcaacaa cgacaaaatc aaacgtgttc
     8761 cgcagagtga tatccgcgag tatattaaca aaggctacca gaaactgatt gaatggtcag
     8821 gtaataatag catccagaaa ggtaatgtgc tgccgcagtg gcatgttatt ccgcatattg
     8881 acaaactgta caaactgctg tgggttccgt ttctgaacga tagcaaactg gaaccggatg
     8941 ttcgttatcc ggttctgaat gaagaatcca aaggttatat tgagggcagc gattacacct
     9001 ataaaaagct gggagataaa gataacctgc cgtataaaac ccgtgttaaa ggtctgacca
     9061 caccgtggtc accgtggaat ccgtttcagg tgattgccga acatgaagaa caagaagtga
     9121 acgttaccgg tagccgtccg agtgttaccg ataaaattga acgtgatggt aaaatggtgt
     9181 aataataac
//
