LOCUS       sequence-227029-        7380 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TcRT-Cas1-Cas2 under pT7lac promoter, encodes TcCRISPR 
            array 2, compatible with SENECA acquisition readout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7380)
  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 7380)
  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_0377_pET30-RT-CRISPR_Tolypothrix_campylonemoides_ARRAY_2
FEATURES             Location/Qualifiers
     source          1..7380
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(409..1224)
                     /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(409..1224)
                     /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     1132..1151
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1216..1235
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1346..1934
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1835..1854
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2088..2105
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2120..2259
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2185..2204)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2345..2367
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2361..2552)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3361..4443)
                     /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     4405..4424
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4444..4521)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4727..4746
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4834..4853
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4834..4852
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4853..4877
                     /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             4892..4914
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(7293..7311)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7307..7354
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ccaggtgttg acttccgcga acgtcctaca gataagggtt tgaggcttat tgatatcttt
       61 ttattgagaa atgattgcag ttattctgac acaatttgac gttccgcgca aaatggttgt
      121 agacttggct caagggaaag ctttgagaac cctgctctgt taaacttctc ccgaagttga
      181 attaatggaa acgtcccgcg gccgctattc ttttgattta taagggattt tgccgatttc
      241 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
      301 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
      361 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
      421 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
      481 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
      541 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
      601 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
      661 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
      721 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
      781 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
      841 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
      901 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
      961 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     1021 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     1081 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     1141 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     1201 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     1261 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1321 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     1381 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1441 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     1501 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1561 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1621 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1681 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     1741 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     1801 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     1861 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     1921 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggtc ttttgctggc
     1981 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2041 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2101 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     2161 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     2221 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     2281 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     2341 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     2401 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     2461 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     2521 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     2581 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     2641 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     2701 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     2761 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     2821 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagatcat tcatgttgtt
     2881 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     2941 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     3001 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     3061 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     3121 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     3181 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagatagt cataagtgcg
     3241 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     3301 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     3361 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3421 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     3481 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     3541 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     3601 acatgagcta tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     3661 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     3721 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     3781 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     3841 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     3901 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccat cttcatggga
     3961 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     4021 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     4081 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     4141 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     4201 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     4261 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     4321 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     4381 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     4441 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     4501 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     4561 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     4621 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     4681 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     4741 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     4801 agaggatcga gatcgatctc gatcccgcga aattaatacg actcactata ggggaattgt
     4861 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca
     4921 tatgtttact atagagcaga ttacttcggc ttggttactt gtgagagccg gctcaagagg
     4981 agccggggtc gatggtatga ctgtcgatct attcgcagca ggagttaacg agcaacttag
     5041 aatattactg aggcagttgc agcaggagag ttatagagcc tctcctgcta agggattctt
     5101 cgtcgcaaag aagtcgggtg gaaagaggct gatcggaatc ccaactgttc gagataggat
     5161 agttcagaga ttacttttgg aagaattgta cttcccccta gaggacacct tcctagactg
     5221 tagttatgct tacagaccgg gtagaaacat ccaacaggcc gtgcagcacc tgtacagcta
     5281 ttatcaatac cagcccaagt ggatcattaa ggctgatatc gctgagttct tcgataatct
     5341 atgctgggct cttttattta cagccctgga agatctccaa ctcgaaccta tacttctaca
     5401 gctgttagag caacagttaa agtccggcat agttattgca ggcaagccaa tctatccagg
     5461 gaaaggtgta ttgcagggcg gggtcttgag tggagcactg gctaacttgt atcttacgtc
     5521 attcgagcga aagtgcctat cctacggtat caaccttgtt agatatggtg acgacttcgc
     5581 tatagcttgc tcgtcctggc ttgaggctaa caggatattg gataagatca ctacatggtt
     5641 aggcgagctg tacctcaacc tacaacctga gaaaacccag atcttcgctc cggacgacga
     5701 atttactttt ctaggttata gattcgctgg cggtgaggtg tatgccccgc ccccacctgt
     5761 aatgacgagg gaaggtgagt gggtcaccaa tgagtccgga ctgccatact tcaggcctaa
     5821 atctcgtcct gttaagttcg tgagtagacc acccaaagcc tgttctattg cttcaccaat
     5881 taagttccca acggccccaa ttagccactt atggcaggag ttcatgacta ccctatatgt
     5941 tacagatcag ggtgcatacc tttctgttaa gaaccaacaa ttccaagttt tctaccaggg
     6001 cgagctgaaa atcaaggtgc ctgctactag agtcaataac attgtaatgt tcggatgctg
     6061 caacgtcagt catggcgctg tcagtatggc cttgaggaga agaataccta ttatgtactt
     6121 atcacagaag ggtagatact tcggccacac cgctgttcag ggcgacgcac gagtggagta
     6181 cctcatgcag caagttaaat gttgcgaaaa tacacaattc acccgacaac aggccgaggc
     6241 tatcgtggct gctaaattac acaactccag aatcttactg atgagactca ataggagaag
     6301 gccgactgag atagcaacac aggccattga cttgatagaa atccttattg actcactgcc
     6361 acaggccgag tccctcgacg ccctaagagg ttacgaggga aaggccgcta ccgtctactt
     6421 tcaggcactc ggctcactat tcactggctt cttcgctttc gacaaacgta ctaaaagacc
     6481 acccacagat cccataaatt ctctgatgtc tctggggtat acccttttgt ctcaacaagt
     6541 attcagtttc attcaatcag taggcttgca tacacatttc ggtaacctac acacaccaag
     6601 ggacaaccac cctgccctcg ttagcgacct gatggaagaa tttcgagccc agatcgtgga
     6661 ctctttcgta gcatatctgg ttaacaagaa gatccttacc cctgaggact tcacaccccc
     6721 agatgagaga ggtggagtgt atctccaggc ttcagctctg aagaaatacc tgaaacactg
     6781 ggaagaaaag ttgcagacag aaactactca cccacatact ggttacaaag ttgcctacag
     6841 aagatgtatt gagctacaag ttagggaata catcgcttgc ctggtcggag aggtcgaagt
     6901 ctatcgacct atggtctgga agttgtagcg tacgttaact ttaagaagga gaacttaaga
     6961 tgttctactt ggtatgctat gacattgtgt cagataatcg aagaaacaaa gtatcaaagc
     7021 tgttggaagc atacggactt agagtccaga agtctgtgtt tgaatgcgtc ctagacgaaa
     7081 agcaatatga gatgttgtcc aagtatttga tgcgtctggt taacaggcga gaggaccaag
     7141 tcaggtttta tccaatgtcc gcccacaaca gatgcaaggt cgctgtgctg ggcacccagc
     7201 ccgagttcgt tgttgacgat gctgcattca tcgtgtgact cgagatccgg ctgctaacaa
     7261 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     7321 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata
//
