LOCUS       sequence-227036-        6346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli codon-optimized FsRT-Cas1-Cas2 under ptetA 
            promoter, encodes FsCRISPR array 2, compatible with SENECA 
            acquisition readout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6346)
  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 6346)
  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_0453_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(opt)_T7term_
            ARRAY2_FaqI
FEATURES             Location/Qualifiers
     source          1..6346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(210..1025)
                     /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(210..1025)
                     /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     933..952
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1017..1036
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1147..1735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1636..1655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1889..1906
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1921..2060
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1986..2005)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2146..2168
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2162..2353)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     RBS             2883..2894
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2901..3524
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2974..2993)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      3569..3640
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3656..3683
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    3698..3716
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3723..3741
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(6025..6043)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6039..6086
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggccgctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
       61 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
      121 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
      181 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
      241 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
      301 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
      361 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
      421 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
      481 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
      541 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
      601 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
      661 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
      721 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
      781 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
      841 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
      901 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
      961 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     1021 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     1081 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1141 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     1201 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     1261 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     1321 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     1381 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     1441 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     1501 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     1561 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     1621 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     1681 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     1741 cagcaacgcg gcctttttac ggttcctggt cttttgctgg ccttttgctc acatgttctt
     1801 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     1861 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     1921 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     1981 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     2041 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2101 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2161 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     2221 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     2281 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     2341 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     2401 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     2461 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     2521 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     2581 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     2641 gactttacga aacacggaaa ccgaagatca ttcatgttgt tgctcaggtc gcagacgttt
     2701 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     2761 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     2821 gggccgccat gccggcggca cgtaagaggt tccaactttc accataatga aacatactag
     2881 agaaagagga gaaatactag atgtccagat tagataaaag taaagtgatt aacagcgcat
     2941 tagagctgct taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc
     3001 taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg
     3061 ccttagccat tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa
     3121 gctggcaaga ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc
     3181 gcgatggagc aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg
     3241 aaaatcaatt agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac
     3301 tcagcgctgt ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg
     3361 ctaaagaaga aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta
     3421 tcgaattatt tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca
     3481 tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc ctaataacac tgatagtgct
     3541 agtgtagatc actactagag ccaggcatca aataaaacga aaggctcagt cgaaagactg
     3601 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
     3661 accttcgggt gggcctttct gcgtttatat actagagtcc ctatcagtga tagagattga
     3721 catccctatc agtgatagag atactgagca ctactagaga agcttgcgga ggagcatgca
     3781 tgtttaccat cgacgagatg ctgagcaaga acaaccaacg tctggcgttc gaacactttg
     3841 cgaccaaaaa cgacggttgc ggcccggacg gtatgcacgt tagcgaactg gagaagtact
     3901 ggcgtatgaa tcacgaccag atcattagcg atctgaagaa ccaggaatac cagccgggta
     3961 ttatcctgat tcgtgagcac atgaataaaa ccggcaaacg ccgtaatatc gcgagcctga
     4021 acgttattga tcgtttcatt acccgtctgc tgagccagaa actgaaccgt tatctggcgc
     4081 cgattttctg cgagaacagc tatgcgtacc aggacagcaa aggcgttatg ccggcggttc
     4141 tgaaagcgaa ggagtacgtt gagctgggta tgcgtcacgt gattgaaatc gatctgaaaa
     4201 actactttga caccattccg ctggaaaacc tgatcccgga gatcgaacgc tacatcaccg
     4261 atgaggcggt tctgcatctg atcaagcaat acctgttttg cgatattagc ttcgagggca
     4321 aaatcagccg taaaacccaa ggtatcgttc aaggtaacgc gatcagcccg atcctgagca
     4381 acctgtacct gaacgacttc gacaaggaac tggacgagag caagctgtgc tggattcgtt
     4441 acgcggacaa tatctacatc tatatggata gctatgagaa ggcgctgctg gtgtatagcg
     4501 agctgaccga gcgtctggag cgtcgcaagc tgaccgttaa caaggagaaa agcggcgttt
     4561 ttgatgtgag cacccgtagc attctgggct acgacatcct gattcgtaac aagaaagtgg
     4621 acgttcgtaa gcatatttac aaaagcgtta atcagtacag caactggcac gatagccgcc
     4681 tggagtttat caacggtcgt taccacatca ccagcgatgg catcctgaat cgccaagact
     4741 tcggcctgct gttcgagaat gagcagaaaa aacactacat cccggttgag gttagcgatc
     4801 agctgaacat ctacggtaat gttaccctgg cgagcaatgt gctgcaaagt ttcagcaacc
     4861 gcgaaatcaa agtgagcttc ttcgacaagt atggtcgtct gattggtagc ttcctgccgg
     4921 aaaaaaccaa gaaaagcgcg gagatcattc tggtgcagag caagaactat ctgaacgaag
     4981 atgtgcgtat ggataccgcg cgccgtatgg aaattgcggg tctgcacaac atccgtgcga
     5041 atctgcgcta ctacgataag aaacacaagg gcgactttaa agagaaggtt gacgcgatta
     5101 gcggctacat tgacgcgctg aaccgtgcgc cgagcgttaa tgatatgatg ctgctggaag
     5161 cgaaagcgcg ccaactgtat tatacctgtt ttaaccaaat cctggaaacc agcgacttcc
     5221 agttcgaaaa gcgtaccaag cgtccgccga aagatgcgat caatgcgtgc attagcttcg
     5281 gcaataccct gctgtataat ctatttgtga atattatctg gaagaagggt ctggacccgc
     5341 gctttggcgt tgttcacgcg agcaacaaac gcaatcaaag cctgaacctg gacttcgcgg
     5401 acatctttaa accgatcgtg attgaccgca ttatcttcac catgatcaat aagaaaatgc
     5461 tgaccctgct gaccgatttt gaaaccagca accagggtgt gtatctgagc cgtgagggca
     5521 agaacatctt cctgcaaatg tacgaggaga agctgaaaag ccgcattacc atcaagggta
     5581 aagagatgag ctattaccag ctgctggaga gcgaagtgca gaattacaag aatttcattc
     5641 tgaccggcga aacctataaa ccgtataagt attattaacg tacgttaact ttaagaagga
     5701 gaacttaaga tgtatgttat cctggtgtac gacatccacc agaagcgtgt tggtaaagcg
     5761 ctgaaaatct gccgtaagta cctgatccac atccaaaaga gcgtgttcga aggcaacatc
     5821 accgagagca aactgaaggc gctgaaagag gaactgggtc acctgattga cacccagatg
     5881 gatagcgtga ttatctacca cctggacagc gttaagtaca ccaaaaagga gcagatcggt
     5941 attgtgcaga gcaccagcaa tgtgatctaa ctcgagatcc ggctgctaac aaagcccgaa
     6001 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     6061 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga taccaggtgc
     6121 ggggaagctt atgttccata gcaaaaagtc ggtcagtctc gtggctgaaa tcatgagttc
     6181 cacaaaatgg ctgaaattca aggaaaatca ggaatctcag aaaaacgatc gaccgacttt
     6241 ttcgataaaa tggttgcaaa aatgagaaaa atctgattta atagaatctg aaaacagcgg
     6301 aaatgctgtt gtcgtacttt acctaaaagg aattgaaacg tcccgc
//
