LOCUS       sequence-227041-        7247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FsRT-Cas1-Cas2 under pT7lac promoter, encodes FsCRISPR 
            array 2 RC, compatible with SENECA acquisition readout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7247)
  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 7247)
  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_0354_pET30-RT-CRISPR_Fusicatenibacter_saccharivorans_ARRAY_2_RC
FEATURES             Location/Qualifiers
     source          1..7247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(402..1217)
                     /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(402..1217)
                     /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     1125..1144
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1209..1228
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1339..1927
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1828..1847
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2081..2098
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2113..2252
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2178..2197)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2338..2360
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2354..2545)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3354..4436)
                     /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     4398..4417
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4437..4514)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4720..4739
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4827..4846
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4827..4845
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4846..4870
                     /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             4885..4907
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(7160..7178)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7174..7221
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ccaggttttg ttcggtgtta tttttgtact catttttaga gaaaataaag aagaatggtt
       61 atggcaaaga aaaaggcagc aatatcatca ccgcttccaa gctctgacct ggttgatctt
      121 tatattagag tttctgtgcc acattggggt ctgaccccat atagcacctt taggtaaagt
      181 acgacgtccc gcggccgcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
      241 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
      301 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      361 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
      421 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
      481 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
      541 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
      601 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
      661 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
      721 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
      781 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
      841 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
      901 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
      961 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     1021 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     1081 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     1141 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     1201 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     1261 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1321 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1381 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1441 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     1501 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1561 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1621 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1681 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1741 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1801 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1861 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1921 atggaaaaac gccagcaacg cggccttttt acggttcctg gtcttttgct ggccttttgc
     1981 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     2041 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     2101 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2161 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     2221 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     2281 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     2341 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     2401 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     2461 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     2521 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     2581 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     2641 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     2701 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     2761 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     2821 tccgcgtttc cagactttac gaaacacgga aaccgaagat cattcatgtt gttgctcagg
     2881 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     2941 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     3001 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     3061 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     3121 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     3181 ctgccggcac ctgtcctacg agttgcatga taaagaagat agtcataagt gcggcgacga
     3241 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     3301 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     3361 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3421 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     3481 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     3541 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     3601 ctatcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     3661 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     3721 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     3781 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     3841 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     3901 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac catcttcatg ggagaaaata
     3961 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     4021 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     4081 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     4141 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     4201 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     4261 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     4321 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     4381 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     4441 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     4501 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     4561 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     4621 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     4681 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     4741 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     4801 cgagatcgat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     4861 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgttc
     4921 actatagacg agatgctatc aaagaacaac caaagattgg ctttcgagca cttcgctact
     4981 aagaacgacg gatgtggccc tgatggtatg cacgtatctg agctggagaa atattggcgt
     5041 atgaatcacg atcagatcat ctccgacttg aagaaccaag agtaccaacc cggcattatt
     5101 ttgattagag aacacatgaa caaaacaggt aagagaagga acattgcatc tctcaacgtg
     5161 atcgacaggt tcataactcg actgctgtcg caaaagttga atagatactt agcccccatt
     5221 ttctgcgaga actcatacgc ttaccaagac tcaaaagggg tgatgcccgc cgtactaaaa
     5281 gccaaggagt acgtcgagtt gggtatgaga cacgttatcg agatagacct caagaattac
     5341 ttcgacacga tcccgttaga gaacttaatc ccagagatcg agcgttacat aacagatgag
     5401 gcagtgctgc acttgattaa acagtactta ttctgtgaca tctcgttcga gggtaagata
     5461 tcacgtaaaa ctcaagggat cgttcagggt aacgcaattt ccccaatact atccaacctc
     5521 tacctgaacg acttcgacaa ggagctggac gagtcgaagc tgtgctggat cagatacgcc
     5581 gacaacatat acatctatat ggacagctac gagaaggccc tactggttta ttccgagctt
     5641 accgagagac ttgagagaag gaaattaacc gttaacaagg agaaatcagg ggtgtttgat
     5701 gttagtaccc gttctatact aggttacgac atcctgatca gaaacaagaa ggttgacgtg
     5761 cgtaaacaca tctacaagtc tgttaaccag tactccaact ggcacgactc tcgattggag
     5821 ttcatcaacg gtcgttacca catcacttcc gacggtatcc tcaatagaca agacttcggt
     5881 ctactgttcg agaatgagca gaagaagcac tacatacctg ttgaggtttc tgaccagttg
     5941 aatatttacg gaaacgttac gctcgccagc aacgttcttc aatcattctc caaccgagag
     6001 atcaaagtgt ccttcttcga caagtacggg agacttattg gatctttcct gcccgagaaa
     6061 actaagaaaa gcgccgagat aatactggtt cagtctaaga actaccttaa cgaggacgtt
     6121 aggatggaca cggcccgacg tatggagatc gccggtttgc ataacatcag agccaacctc
     6181 agatactacg acaagaagca caaaggtgac tttaaggaaa aggtagacgc catttctggc
     6241 tatattgacg ccctcaatag agcaccatcc gtgaacgaca tgatgctctt agaggctaaa
     6301 gctcgtcagc tgtactacac atgcttcaac cagatcttag agacatccga ctttcagttc
     6361 gagaagcgta ccaagcgacc accaaaagac gccatcaacg cctgtatttc attcggcaac
     6421 acattactgt acaacttgtt tgtgaatatc atttggaaga agggacttga tccaaggttc
     6481 ggggttgttc acgccagcaa caagcgtaat cagtccctta acctggattt tgctgacatc
     6541 ttcaagccta tagtcataga taggatcatc ttcacaatga tcaacaagaa gatgctgacc
     6601 ttgttgacgg acttcgagac ttctaaccag ggagtttacc tttcccgaga ggggaagaac
     6661 atcttcttgc agatgtacga agaaaagctc aaatccagaa tcactatcaa gggaaaggag
     6721 atgagttact atcagttgct agaatcagag gtgcagaact acaagaactt catcctgacc
     6781 ggcgagacat acaagccgta caagtactac tagcgtacgt taactttaag aaggagaact
     6841 taagatgtat gtcattctag tttacgacat tcaccagaag cgagtaggta aggctctaaa
     6901 gatttgtagg aagtacctga tccacatcca gaagtcagtt ttcgaaggca acataacgga
     6961 gagtaagcta aaggccttaa aagaggagct ggggcacctg atcgacactc agatggactc
     7021 ggtaattatc taccacctgg actccgtgaa gtacactaag aaggagcaga tcggcatcgt
     7081 ccaatcgaca tccaatgtca tttgactcga gatccggctg ctaacaaagc ccgaaaggaa
     7141 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     7201 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggata
//
