LOCUS       sequence-361528-        6616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes aTc inducible FsRT-Cas1–Cas2 expression cassette and 
            FsCRISPR Array 2 transcribed by BBa_J23109.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6616)
  AUTHORS   Schmidt F, Zimmermann J, Tanna T, Farouni R, Conway T, Macpherson 
            AJ, Platt RJ
  TITLE     Noninvasive assessment of gut function using transcriptional 
            recording sentinel cells.
  JOURNAL   Science. 2022 May 13;376(6594):eabm6038. doi: 
            10.1126/science.abm6038. Epub 2022 May 13.
  PUBMED    35549411
REFERENCE   2  (bases 1 to 6616)
  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"; journalName: "Science."; 
            date: "2022-05-13"; volume: "376(6594):eabm6038. doi"; pages: " 
            10.1126/science.abm6038. Epub 2022 May 13"
COMMENT     Sequence Label: 
            pFS_1112_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(opt)-3xTerm_
            J23109-Leader-ARRAY2-DR2-FaqI_Term_NotI
FEATURES             Location/Qualifiers
     source          1..6616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(209..1024)
                     /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(209..1024)
                     /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     932..951
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1016..1035
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1146..1734
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1635..1654
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1888..1905
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1920..2059
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1985..2004)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2145..2167
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2161..2352)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     RBS             2882..2893
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2900..3523
                     /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(2973..2992)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      3568..3639
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3655..3682
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    3697..3715
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3722..3740
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      complement(6030..6073)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(6116..6134)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6130..6177
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        6202..6236
                     /label=BBa_J23109
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.04"
ORIGIN
        1 gccgctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
       61 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
      121 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
      181 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
      241 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
      301 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
      361 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
      421 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
      481 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
      541 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
      601 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
      661 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
      721 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
      781 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
      841 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
      901 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
      961 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     1021 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     1081 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1141 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1201 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1261 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1321 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1381 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1441 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1501 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1561 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1621 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     1681 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     1741 agcaacgcgg cctttttacg gttcctggtc ttttgctggc cttttgctca catgttcttt
     1801 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     1861 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     1921 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     1981 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     2041 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2101 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     2161 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     2221 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     2281 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     2341 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     2401 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     2461 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     2521 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     2581 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     2641 actttacgaa acacggaaac cgaagatcat tcatgttgtt gctcaggtcg cagacgtttt
     2701 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     2761 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     2821 ggccgccatg ccggcggcac gtaagaggtt ccaactttca ccataatgaa acatactaga
     2881 gaaagaggag aaatactaga tgtccagatt agataaaagt aaagtgatta acagcgcatt
     2941 agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct
     3001 aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc
     3061 cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag
     3121 ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg
     3181 cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga
     3241 aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact
     3301 cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc
     3361 taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac gacaagctat
     3421 cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat
     3481 atgcggatta gaaaaacaac ttaaatgtga aagtgggtcc taataacact gatagtgcta
     3541 gtgtagatca ctactagagc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     3601 gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca
     3661 ccttcgggtg ggcctttctg cgtttatata ctagagtccc tatcagtgat agagattgac
     3721 atccctatca gtgatagaga tactgagcac tactagagaa gcttgcggag gagcatgcat
     3781 gtttaccatc gacgagatgc tgagcaagaa caaccaacgt ctggcgttcg aacactttgc
     3841 gaccaaaaac gacggttgcg gcccggacgg tatgcacgtt agcgaactgg agaagtactg
     3901 gcgtatgaat cacgaccaga tcattagcga tctgaagaac caggaatacc agccgggtat
     3961 tatcctgatt cgtgagcaca tgaataaaac cggcaaacgc cgtaatatcg cgagcctgaa
     4021 cgttattgat cgtttcatta cccgtctgct gagccagaaa ctgaaccgtt atctggcgcc
     4081 gattttctgc gagaacagct atgcgtacca ggacagcaaa ggcgttatgc cggcggttct
     4141 gaaagcgaag gagtacgttg agctgggtat gcgtcacgtg attgaaatcg atctgaaaaa
     4201 ctactttgac accattccgc tggaaaacct gatcccggag atcgaacgct acatcaccga
     4261 tgaggcggtt ctgcatctga tcaagcaata cctgttttgc gatattagct tcgagggcaa
     4321 aatcagccgt aaaacccaag gtatcgttca aggtaacgcg atcagcccga tcctgagcaa
     4381 cctgtacctg aacgacttcg acaaggaact ggacgagagc aagctgtgct ggattcgtta
     4441 cgcggacaat atctacatct atatggatag ctatgagaag gcgctgctgg tgtatagcga
     4501 gctgaccgag cgtctggagc gtcgcaagct gaccgttaac aaggagaaaa gcggcgtttt
     4561 tgatgtgagc acccgtagca ttctgggcta cgacatcctg attcgtaaca agaaagtgga
     4621 cgttcgtaag catatttaca aaagcgttaa tcagtacagc aactggcacg atagccgcct
     4681 ggagtttatc aacggtcgtt accacatcac cagcgatggc atcctgaatc gccaagactt
     4741 cggcctgctg ttcgagaatg agcagaaaaa acactacatc ccggttgagg ttagcgatca
     4801 gctgaacatc tacggtaatg ttaccctggc gagcaatgtg ctgcaaagtt tcagcaaccg
     4861 cgaaatcaaa gtgagcttct tcgacaagta tggtcgtctg attggtagct tcctgccgga
     4921 aaaaaccaag aaaagcgcgg agatcattct ggtgcagagc aagaactatc tgaacgaaga
     4981 tgtgcgtatg gataccgcgc gccgtatgga aattgcgggt ctgcacaaca tccgtgcgaa
     5041 tctgcgctac tacgataaga aacacaaggg cgactttaaa gagaaggttg acgcgattag
     5101 cggctacatt gacgcgctga accgtgcgcc gagcgttaat gatatgatgc tgctggaagc
     5161 gaaagcgcgc caactgtatt atacctgttt taaccaaatc ctggaaacca gcgacttcca
     5221 gttcgaaaag cgtaccaagc gtccgccgaa agatgcgatc aatgcgtgca ttagcttcgg
     5281 caataccctg ctgtataatc tatttgtgaa tattatctgg aagaagggtc tggacccgcg
     5341 ctttggcgtt gttcacgcga gcaacaaacg caatcaaagc ctgaacctgg acttcgcgga
     5401 catctttaaa ccgatcgtga ttgaccgcat tatcttcacc atgatcaata agaaaatgct
     5461 gaccctgctg accgattttg aaaccagcaa ccagggtgtg tatctgagcc gtgagggcaa
     5521 gaacatcttc ctgcaaatgt acgaggagaa gctgaaaagc cgcattacca tcaagggtaa
     5581 agagatgagc tattaccagc tgctggagag cgaagtgcag aattacaaga atttcattct
     5641 gaccggcgaa acctataaac cgtataagta ttattaacgt acgttaactt taagaaggag
     5701 aacttaagat gtatgttatc ctggtgtacg acatccacca gaagcgtgtt ggtaaagcgc
     5761 tgaaaatctg ccgtaagtac ctgatccaca tccaaaagag cgtgttcgaa ggcaacatca
     5821 ccgagagcaa actgaaggcg ctgaaagagg aactgggtca cctgattgac acccagatgg
     5881 atagcgtgat tatctaccac ctggacagcg ttaagtacac caaaaaggag cagatcggta
     5941 ttgtgcagag caccagcaat gtgatctaac tcgagtagcc tagcataacc ccgcggggcc
     6001 tcttcggggg tctcgcgggg ttttttgcta taaaacgaaa ggctcagtcg aaagactggg
     6061 cctttcgttt tatctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     6121 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     6181 aaaggaggaa ctatatccgg atttacagct agctcagtcc tagggactgt gctagctcta
     6241 gtagtggcgg ggaagcttat gttccatagc aaaaagtcgg tcagtctcgt ggctgaaatc
     6301 atgagttcca caaaatggct gaaattcaag gaaaatcagg aatctcagaa aaacgatcga
     6361 ccgacttttt cgataaaatg gttgcaaaaa tgagaaaaat ctgatttaat agaatctgaa
     6421 aacagcggaa atgctgttgt cgtactttac ctaaaaggaa ttgaaacgtc cccgccaggt
     6481 tgaatccgat atttggaggt acgatggaac agtctgggtg ggattgagaa gagaaaagaa
     6541 aaccgccgat cctgtccacc gcattactgc aaggtagtgg acaagaccgg cggtcttaag
     6601 ttttttggct gaagcg
//
