LOCUS       sequence-354257-        5368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pACYC Duet-1 containing full Type III-E system Candidatus "Scalindua
            brodae" CRISPR array.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5368)
  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 5368)
  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: pTU387
FEATURES             Location/Qualifiers
     source          1..5368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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             1724..1768
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1806..1824)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1820..1867
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2088..2747)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2662..2681
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2748..2850)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3242..3259)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3376..3921)
                     /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(3456..3475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4146..5228)
                     /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     5190..5209
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5229..5306)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5352..5368,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tggcagatct caattggata tccagacaaa cggtttgcga agaaatacgc gacagggtga
      361 ttggaccgta acctcatgat tatatgattg atacacgatt taaccctgac ttgccggttt
      421 ttgaaaaagt tcgcaaaccc tgttttgctt catgaagtga gttgggtttg cgaaaaaagg
      481 ttattacagc ctgatatcta agtagaagag taccggtatt gaagaccaaa gttgctgcgt
      541 atggcggtcc ggttgtcctt gctttcgcaa ggattccaat actggaatcc tcccgaaagg
      601 gaggtcgcaa aaggccgttt ttcgaaaacc atagtttcat acaaaccggc gatgaggttt
      661 gcgaactttt tgattgtagt aagtattatt aaaataatgg cttaatattt ttggtatata
      721 caattctcaa ctttttcacc ttgccggaaa tgaggtttgc gaaattttag agagccgcat
      781 atctatatta tttacaatca gttacaaaat ggccccttct cgccatatac gtaacctcag
      841 agttgttgga gggttatgaa acaagagaag gacttaatgt cacggtaccc aattttctgc
      901 cccggactcc acggctgtta ctagaggtta tgaaacaaga gaaggactta atgtcacggt
      961 acagtttcct gttttttttg ctccctaacg ctactttgaa tgttatgaaa caagagaagg
     1021 acttaatgtc acggtacaat tatcatttgg acagcttccc tcattatttc gaggtcgtta
     1081 tgaaacaaga gaaggactta atgtcacggt acgaaaaaaa aaagtaaagt tcaggggcaa
     1141 gtgccaaagt tatgaaacaa gagaaggact taatgtcacg gtaccccttt gcttcttctc
     1201 tagtgtttct atccatgttt gtgttatgaa acaagagaag gacttaatgt cacggtactt
     1261 acgaagtatc tccgtacgaa ccttttcact gtgttatgaa acaagagaag gacttaatgt
     1321 cacggtacag aattggtatt atttttccca gtgtaataat accgttatga aacaagagaa
     1381 ggacttaatg tcacggtaca ccatttttgt cattatttat tgtcatgtta gaaagttatg
     1441 aaacaagaga aggacttaat gtcacggtac tcttcagcaa ttacttcttt acgaagagat
     1501 aactttgtta tgaaacaaga gaaggactta atgtcacggt acaaaatctc aagcctcaag
     1561 catatactca aaatcattag ttatgaaaca agagaaggac ttaatgtcac ggtacatcat
     1621 taccatccat atgttctgat gactgtctct gctgtagtta tgaaacaaga gaagtactta
     1681 ggtttgattg aagatcgctg acgtcggtac cctcgagtct ggtaaagaaa ccgctgctgc
     1741 gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa cctaggctgc
     1801 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1861 ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa
     1921 taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac
     1981 gaccgggtcg aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag
     2041 gcgtagcaac caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc
     2101 tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
     2161 caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
     2221 tatttgccca tagtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
     2281 aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
     2341 ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
     2401 aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
     2461 tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
     2521 gccatacgga actccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
     2581 taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
     2641 gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
     2701 cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta
     2761 gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg
     2821 tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccaaaa gttggcccag
     2881 ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc
     2941 acaggtattt attcggcgca aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt
     3001 atgatggtgt ttttgaggtg ctccagtggc ttctgtttct atcagctgtc cctcctgttc
     3061 agctactgac ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctagcggag
     3121 tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca
     3181 ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt
     3241 cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg
     3301 aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc
     3361 cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg
     3421 ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3481 cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt
     3541 atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca gttcgctcca
     3601 agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta tccggtaact
     3661 atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca gccactggta
     3721 attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca
     3781 agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag
     3841 agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca agagattacg
     3901 cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata tttctagatt
     3961 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg
     4021 taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     4081 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4141 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4201 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     4261 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     4321 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     4381 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     4441 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     4501 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     4561 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     4621 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     4681 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     4741 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     4801 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     4861 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     4921 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     4981 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     5041 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     5101 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     5161 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     5221 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     5281 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     5341 cattaggaaa ttaatacgac tcactata
//
