LOCUS       sequence-227101-        7379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TcRT-Cas1-Cas2 under pT7lac promoter, encodes TcCRISPR 
            array 3, compatible with SENECA acquisition readout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7379)
  AUTHORS   Schmidt F, Cherepkova MY, Platt RJ
  TITLE     Transcriptional recording by CRISPR spacer acquisition from RNA.
  JOURNAL   Nature. 2018 Oct;562(7727):380-385. doi: 10.1038/s41586-018-0569-1. 
            Epub 2018 Oct 3.
  PUBMED    30283135
REFERENCE   2  (bases 1 to 7379)
  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: 
            "10.1038/s41586-018-0569-1"; journalName: "Nature"; date: "2018-10";
            volume: "562"; issue: "7727"; pages: "380-385"
COMMENT     Sequence Label: 
            pFS_0379_pET30-RT-CRISPR_Tolypothrix_campylonemoides_ARRAY_3
FEATURES             Location/Qualifiers
     source          1..7379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(408..1223)
                     /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(408..1223)
                     /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     1131..1150
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1215..1234
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1345..1933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1834..1853
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2087..2104
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2119..2258
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2184..2203)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2344..2366
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2360..2551)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3360..4442)
                     /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     4404..4423
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4443..4520)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4726..4745
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4833..4852
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4833..4851
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4852..4876
                     /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             4891..4913
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(7292..7310)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7306..7353
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ccaggtttct atcccacaaa ttagcttcct cacaaaggtt tgaggtactt tggctggaga
       61 tttgttgaga gtaattcgca tttgtttgga cgaaaatttg catcccacaa aaaagcgctg
      121 tagaattatt tcagggtaag gcttctagga gctggtcttt aaacttcttc ggaagttgaa
      181 ttaatggaaa cgtcccgcgg ccgctattct tttgatttat aagggatttt gccgatttcg
      241 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
      301 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
      361 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
      421 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
      481 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
      541 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
      601 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
      661 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
      721 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
      781 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
      841 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
      901 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
      961 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     1021 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     1081 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     1141 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     1201 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     1261 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1321 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1381 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1441 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     1501 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1561 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1621 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1681 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1741 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     1801 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1861 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1921 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggtct tttgctggcc
     1981 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2041 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     2101 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2161 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     2221 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     2281 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     2341 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     2401 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     2461 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     2521 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     2581 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     2641 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     2701 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     2761 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     2821 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagatcatt catgttgttg
     2881 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     2941 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     3001 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     3061 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     3121 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     3181 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagatagtc ataagtgcgg
     3241 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     3301 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     3361 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3421 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     3481 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     3541 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     3601 catgagctat cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc
     3661 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     3721 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     3781 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     3841 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     3901 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccatc ttcatgggag
     3961 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     4021 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     4081 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     4141 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     4201 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     4261 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     4321 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     4381 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     4441 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     4501 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     4561 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     4621 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     4681 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     4741 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     4801 gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     4861 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
     4921 atgtttacta tagagcagat tacttcggct tggttacttg tgagagccgg ctcaagagga
     4981 gccggggtcg atggtatgac tgtcgatcta ttcgcagcag gagttaacga gcaacttaga
     5041 atattactga ggcagttgca gcaggagagt tatagagcct ctcctgctaa gggattcttc
     5101 gtcgcaaaga agtcgggtgg aaagaggctg atcggaatcc caactgttcg agataggata
     5161 gttcagagat tacttttgga agaattgtac ttccccctag aggacacctt cctagactgt
     5221 agttatgctt acagaccggg tagaaacatc caacaggccg tgcagcacct gtacagctat
     5281 tatcaatacc agcccaagtg gatcattaag gctgatatcg ctgagttctt cgataatcta
     5341 tgctgggctc ttttatttac agccctggaa gatctccaac tcgaacctat acttctacag
     5401 ctgttagagc aacagttaaa gtccggcata gttattgcag gcaagccaat ctatccaggg
     5461 aaaggtgtat tgcagggcgg ggtcttgagt ggagcactgg ctaacttgta tcttacgtca
     5521 ttcgagcgaa agtgcctatc ctacggtatc aaccttgtta gatatggtga cgacttcgct
     5581 atagcttgct cgtcctggct tgaggctaac aggatattgg ataagatcac tacatggtta
     5641 ggcgagctgt acctcaacct acaacctgag aaaacccaga tcttcgctcc ggacgacgaa
     5701 tttacttttc taggttatag attcgctggc ggtgaggtgt atgccccgcc cccacctgta
     5761 atgacgaggg aaggtgagtg ggtcaccaat gagtccggac tgccatactt caggcctaaa
     5821 tctcgtcctg ttaagttcgt gagtagacca cccaaagcct gttctattgc ttcaccaatt
     5881 aagttcccaa cggccccaat tagccactta tggcaggagt tcatgactac cctatatgtt
     5941 acagatcagg gtgcatacct ttctgttaag aaccaacaat tccaagtttt ctaccagggc
     6001 gagctgaaaa tcaaggtgcc tgctactaga gtcaataaca ttgtaatgtt cggatgctgc
     6061 aacgtcagtc atggcgctgt cagtatggcc ttgaggagaa gaatacctat tatgtactta
     6121 tcacagaagg gtagatactt cggccacacc gctgttcagg gcgacgcacg agtggagtac
     6181 ctcatgcagc aagttaaatg ttgcgaaaat acacaattca cccgacaaca ggccgaggct
     6241 atcgtggctg ctaaattaca caactccaga atcttactga tgagactcaa taggagaagg
     6301 ccgactgaga tagcaacaca ggccattgac ttgatagaaa tccttattga ctcactgcca
     6361 caggccgagt ccctcgacgc cctaagaggt tacgagggaa aggccgctac cgtctacttt
     6421 caggcactcg gctcactatt cactggcttc ttcgctttcg acaaacgtac taaaagacca
     6481 cccacagatc ccataaattc tctgatgtct ctggggtata cccttttgtc tcaacaagta
     6541 ttcagtttca ttcaatcagt aggcttgcat acacatttcg gtaacctaca cacaccaagg
     6601 gacaaccacc ctgccctcgt tagcgacctg atggaagaat ttcgagccca gatcgtggac
     6661 tctttcgtag catatctggt taacaagaag atccttaccc ctgaggactt cacaccccca
     6721 gatgagagag gtggagtgta tctccaggct tcagctctga agaaatacct gaaacactgg
     6781 gaagaaaagt tgcagacaga aactactcac ccacatactg gttacaaagt tgcctacaga
     6841 agatgtattg agctacaagt tagggaatac atcgcttgcc tggtcggaga ggtcgaagtc
     6901 tatcgaccta tggtctggaa gttgtagcgt acgttaactt taagaaggag aacttaagat
     6961 gttctacttg gtatgctatg acattgtgtc agataatcga agaaacaaag tatcaaagct
     7021 gttggaagca tacggactta gagtccagaa gtctgtgttt gaatgcgtcc tagacgaaaa
     7081 gcaatatgag atgttgtcca agtatttgat gcgtctggtt aacaggcgag aggaccaagt
     7141 caggttttat ccaatgtccg cccacaacag atgcaaggtc gctgtgctgg gcacccagcc
     7201 cgagttcgtt gttgacgatg ctgcattcat cgtgtgactc gagatccggc tgctaacaaa
     7261 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     7321 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggata
//
