LOCUS       sequence-354251-        5857 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pACYC containing Candidatus "Scalindua brodae" TPR-CHAT H585A C627A 
            (codon-optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5857)
  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 5857)
  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: pTU425
FEATURES             Location/Qualifiers
     source          1..5857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     724..743
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(763..840)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     886..905
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        886..904
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    905..929
                     /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             3133..3156
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3197..3215)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3211..3258
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3479..4138)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4053..4072
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4139..4241)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4633..4650)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4767..5312)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4847..4866)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5537..5857,1..762))
                     /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"
ORIGIN
        1 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
       61 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
      121 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
      181 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
      241 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
      301 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
      361 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
      421 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
      481 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
      541 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
      601 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
      661 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
      721 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
      781 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
      841 cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata cgactcacta
      901 taggggaatt gtgagcggat aacaattccc ctgtagaaat aattttgttt aactttaata
      961 aggagatata ccatgaacaa caccgaagaa aacatcgatc gtattcaaga accgacgcgt
     1021 gaagatattg atcgtaaaga agcagaacgt ctgctggatg aagcatttaa tccgcgtacc
     1081 aaaccggtgg atcgcaaaaa aatcattaat agcgcactga aaattctgat cggcctgtac
     1141 aaagagaaaa aagacgatct gaccagcgca agctttatta gcattgcacg tgcctattat
     1201 ctggtgagca ttaccattct gccgaaaggc accaccattc cggaaaaaaa gaaagaagca
     1261 ctgcgcaaag gcatcgaatt tattgatcgc gcaatcaaca agtttaacgg cagcattctg
     1321 gatagccagc gtgcatttcg tattaaaagc gttctgagca ttgagttcaa tcgtatcgat
     1381 cgtgaaaaat gcgacaacat caaactgaaa aacctgctga acgaagccgt tgataaaggt
     1441 tgtaccgatt ttgataccta tgagtgggat attcagattg ccattcgtct gtgtgaactg
     1501 ggtgttgata tggaaggtca ttttgacaac ctgatcaaaa gcaacaaagc caacgatctg
     1561 cagaaagcca aagcctatta cttcatcaaa aaggatgacc ataaggccaa agaacacatg
     1621 gataaatgta ccgcaagcct gaaatatacc ccgtgtagtc atcgtctgtg ggatgaaacc
     1681 gttggtttta ttgaacgtct gaaaggtgat agcagcaccc tgtggcgtga ttttgcaatt
     1741 aaaacctatc gtagctgccg tgtgcaagaa aaagaaaccg gtacactgcg tctgcgttgg
     1801 tattggagcc gtcatcgtgt tctgtatgat atggcatttc tggcagttaa agaacaggca
     1861 gatgatgaag aaccggatgt taatgttaaa caggccaaaa tcaaaaagct ggccgaaatt
     1921 agcgatagcc tgaaaagccg ttttagcctg cgtctgagcg atatggaaaa aatgccgaaa
     1981 agtgatgatg aaagcaacca cgagttcaaa aagtttctgg acaaatgtgt taccgcctat
     2041 caggatggtt atgtgattaa tcgtagcgag gataaagaag gtcagggcga aaacaaaagc
     2101 accaccagta aacagccgga accgcgtccg caggcaaaac tgctggaact gacccaggtt
     2161 ccggaaggtt gggttgttgt tcacttttat ctgaataaac tggaaggtat gggcaacgcc
     2221 attgtgtttg ataaatgtgc aaatagctgg cagtacaaag aatttcagta taaagaactg
     2281 tttgaagtgt tcctgacctg gcaggcaaac tataatcttt acaaagaaaa cgcagccgaa
     2341 catctggtta ccctgtgtaa aaagattggt gaaaccatgc cgtttctgtt ctgcgataac
     2401 tttattccga atggtaaaga tgttctgttt gtgccgcatg attttctgca tcgtctgccg
     2461 ctgcatggta gcattgagaa taaaacaaat ggcaagctgt tcctggaaaa tcatagctgt
     2521 tgttatctgc ctgcatggtc atttgcaagc gaaaaagaag caagcaccag cgacgaatat
     2581 gttctgctga aaaatttcga tcagggccat tttgaaaccc tgcagaataa tcagatttgg
     2641 ggcacccaga gcgttaaaga tggtgcaagc agtgatgatc tggaaaacat tcgtaacaat
     2701 ccgcgtctgc tgaccattct gtgtgcgggt gaagcaaata tgagcaatcc gtttcgtagc
     2761 atgctgaaac tggcaaatgg tggtattacc tatctggaaa ttctgaatag cgtgaaaggc
     2821 ctgaaaggta gccaggttat tctgggtgca gcggaaaccg atctggttcc gcctctgagt
     2881 gatgttatgg atgaacatta tagcgttgca accgcactgc tgctgattgg tgcagcgggt
     2941 gttgttggca ccatgtggaa agttcgtagc aataaaacga aaagcctgat cgagtggaag
     3001 ctggaaaata tcgaatataa actgaacgag tggcagaaag aaacaggtgg tgcagcatat
     3061 aaagatcatc cgcctacctt ttatcgtagc attgcctttc gtagtattgg ttttccgtta
     3121 ggtggtagcg gtcatcacca tcaccaccat catcattaat ctactagcgc agcttaatta
     3181 acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     3241 gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt
     3301 ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc
     3361 cctgaaccga cgaccgggtc gaatttgctt tcgaatttct gccattcatc cgcttattat
     3421 cacttattca ggcgtagcaa ccaggcgttt aagggcacca ataactgcct taaaaaaatt
     3481 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat
     3541 ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc
     3601 cttgcgtata atatttgccc atagtgaaaa cgggggcgaa gaagttgtcc atattggcca
     3661 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct
     3721 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat
     3781 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt
     3841 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac
     3901 cgtctttcat tgccatacgg aactccggat gagcattcat caggcgggca agaatgtgaa
     3961 taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat
     4021 ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt
     4081 ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt
     4141 tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta
     4201 tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaaa
     4261 agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt
     4321 gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttac
     4381 tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc tatcagctgt
     4441 ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc cggacatcag
     4501 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc
     4561 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat
     4621 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga
     4681 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga
     4741 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac
     4801 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg
     4861 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc
     4921 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc
     4981 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt
     5041 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc
     5101 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa
     5161 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt
     5221 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc
     5281 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat
     5341 atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     5401 gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg
     5461 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5521 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5581 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5641 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5701 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5761 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     5821 ccgcaccaac gcgcagcccg gactcggtaa tggcgcg
//
