LOCUS       sequence-361531-        6608 bp    DNA     circular SYN 14-APR-2025
DEFINITION  encodes aTc inducible FsRT-Cas1–Cas2 and transcriptionally insulated
            FsCRISPR Array 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6608)
  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 6608)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_1061_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(opt)-3xTerm_
            GGC(BbsI)-Leader-ARRAY2-DR2-FaqI_Term_NotI
FEATURES             Location/Qualifiers
     source          1..6608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(216..1031)
                     /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(216..1031)
                     /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     939..958
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1023..1042
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1153..1741
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1642..1661
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1895..1912
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1927..2066
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1992..2011)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2152..2174
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2168..2359)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     RBS             2889..2900
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2907..3530
                     /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(2980..2999)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      3575..3646
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3662..3689
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    3704..3722
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3729..3747
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      complement(6037..6080)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(6123..6141)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6137..6184
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tgaagcggcc gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
       61 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
      121 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      181 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
      241 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
      301 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
      361 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
      421 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
      481 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
      541 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
      601 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
      661 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
      721 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
      781 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
      841 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
      901 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
      961 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     1021 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     1081 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1141 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1201 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1261 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     1321 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     1381 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     1441 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     1501 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     1561 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     1621 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     1681 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     1741 aaacgccagc aacgcggcct ttttacggtt cctggtcttt tgctggcctt ttgctcacat
     1801 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     1861 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     1921 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     1981 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     2041 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     2101 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2161 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     2221 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     2281 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     2341 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     2401 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     2461 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     2521 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     2581 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     2641 tttccagact ttacgaaaca cggaaaccga agatcattca tgttgttgct caggtcgcag
     2701 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     2761 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     2821 cccgtggggc cgccatgccg gcggcacgta agaggttcca actttcacca taatgaaaca
     2881 tactagagaa agaggagaaa tactagatgt ccagattaga taaaagtaaa gtgattaaca
     2941 gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt aaactcgccc
     3001 agaagctagg tgtagagcag cctacattgt attggcatgt aaaaaataag cgggctttgc
     3061 tcgacgcctt agccattgag atgttagata ggcaccatac tcacttttgc cctttagaag
     3121 gggaaagctg gcaagatttt ttacgtaata acgctaaaag ttttagatgt gctttactaa
     3181 gtcatcgcga tggagcaaaa gtacatttag gtacacggcc tacagaaaaa cagtatgaaa
     3241 ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt ttcactagag aatgcattat
     3301 atgcactcag cgctgtgggg cattttactt taggttgcgt attggaagat caagagcatc
     3361 aagtcgctaa agaagaaagg gaaacaccta ctactgatag tatgccgcca ttattacgac
     3421 aagctatcga attatttgat caccaaggtg cagagccagc cttcttattc ggccttgaat
     3481 tgatcatatg cggattagaa aaacaactta aatgtgaaag tgggtcctaa taacactgat
     3541 agtgctagtg tagatcacta ctagagccag gcatcaaata aaacgaaagg ctcagtcgaa
     3601 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac
     3661 tggctcacct tcgggtgggc ctttctgcgt ttatatacta gagtccctat cagtgataga
     3721 gattgacatc cctatcagtg atagagatac tgagcactac tagagaagct tgcggaggag
     3781 catgcatgtt taccatcgac gagatgctga gcaagaacaa ccaacgtctg gcgttcgaac
     3841 actttgcgac caaaaacgac ggttgcggcc cggacggtat gcacgttagc gaactggaga
     3901 agtactggcg tatgaatcac gaccagatca ttagcgatct gaagaaccag gaataccagc
     3961 cgggtattat cctgattcgt gagcacatga ataaaaccgg caaacgccgt aatatcgcga
     4021 gcctgaacgt tattgatcgt ttcattaccc gtctgctgag ccagaaactg aaccgttatc
     4081 tggcgccgat tttctgcgag aacagctatg cgtaccagga cagcaaaggc gttatgccgg
     4141 cggttctgaa agcgaaggag tacgttgagc tgggtatgcg tcacgtgatt gaaatcgatc
     4201 tgaaaaacta ctttgacacc attccgctgg aaaacctgat cccggagatc gaacgctaca
     4261 tcaccgatga ggcggttctg catctgatca agcaatacct gttttgcgat attagcttcg
     4321 agggcaaaat cagccgtaaa acccaaggta tcgttcaagg taacgcgatc agcccgatcc
     4381 tgagcaacct gtacctgaac gacttcgaca aggaactgga cgagagcaag ctgtgctgga
     4441 ttcgttacgc ggacaatatc tacatctata tggatagcta tgagaaggcg ctgctggtgt
     4501 atagcgagct gaccgagcgt ctggagcgtc gcaagctgac cgttaacaag gagaaaagcg
     4561 gcgtttttga tgtgagcacc cgtagcattc tgggctacga catcctgatt cgtaacaaga
     4621 aagtggacgt tcgtaagcat atttacaaaa gcgttaatca gtacagcaac tggcacgata
     4681 gccgcctgga gtttatcaac ggtcgttacc acatcaccag cgatggcatc ctgaatcgcc
     4741 aagacttcgg cctgctgttc gagaatgagc agaaaaaaca ctacatcccg gttgaggtta
     4801 gcgatcagct gaacatctac ggtaatgtta ccctggcgag caatgtgctg caaagtttca
     4861 gcaaccgcga aatcaaagtg agcttcttcg acaagtatgg tcgtctgatt ggtagcttcc
     4921 tgccggaaaa aaccaagaaa agcgcggaga tcattctggt gcagagcaag aactatctga
     4981 acgaagatgt gcgtatggat accgcgcgcc gtatggaaat tgcgggtctg cacaacatcc
     5041 gtgcgaatct gcgctactac gataagaaac acaagggcga ctttaaagag aaggttgacg
     5101 cgattagcgg ctacattgac gcgctgaacc gtgcgccgag cgttaatgat atgatgctgc
     5161 tggaagcgaa agcgcgccaa ctgtattata cctgttttaa ccaaatcctg gaaaccagcg
     5221 acttccagtt cgaaaagcgt accaagcgtc cgccgaaaga tgcgatcaat gcgtgcatta
     5281 gcttcggcaa taccctgctg tataatctat ttgtgaatat tatctggaag aagggtctgg
     5341 acccgcgctt tggcgttgtt cacgcgagca acaaacgcaa tcaaagcctg aacctggact
     5401 tcgcggacat ctttaaaccg atcgtgattg accgcattat cttcaccatg atcaataaga
     5461 aaatgctgac cctgctgacc gattttgaaa ccagcaacca gggtgtgtat ctgagccgtg
     5521 agggcaagaa catcttcctg caaatgtacg aggagaagct gaaaagccgc attaccatca
     5581 agggtaaaga gatgagctat taccagctgc tggagagcga agtgcagaat tacaagaatt
     5641 tcattctgac cggcgaaacc tataaaccgt ataagtatta ttaacgtacg ttaactttaa
     5701 gaaggagaac ttaagatgta tgttatcctg gtgtacgaca tccaccagaa gcgtgttggt
     5761 aaagcgctga aaatctgccg taagtacctg atccacatcc aaaagagcgt gttcgaaggc
     5821 aacatcaccg agagcaaact gaaggcgctg aaagaggaac tgggtcacct gattgacacc
     5881 cagatggata gcgtgattat ctaccacctg gacagcgtta agtacaccaa aaaggagcag
     5941 atcggtattg tgcagagcac cagcaatgtg atctaactcg agtagcctag cataaccccg
     6001 cggggcctct tcgggggtct cgcggggttt tttgctataa aacgaaaggc tcagtcgaaa
     6061 gactgggcct ttcgttttat ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     6121 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     6181 tttgctgaaa ggaggaacta tatccggatg tcttcatggt agtaccaaga tacgaagaca
     6241 tagtggcggg gaagcttatg ttccatagca aaaagtcggt cagtctcgtg gctgaaatca
     6301 tgagttccac aaaatggctg aaattcaagg aaaatcagga atctcagaaa aacgatcgac
     6361 cgactttttc gataaaatgg ttgcaaaaat gagaaaaatc tgatttaata gaatctgaaa
     6421 acagcggaaa tgctgttgtc gtactttacc taaaaggaat tgaaacgtcc ccgccaggtt
     6481 gaatccgata tttggaggta cgatggaaca gtctgggtgg gattgagaag agaaaagaaa
     6541 accgccgatc ctgtccaccg cattactgca aggtagtgga caagaccggc ggtcttaagt
     6601 tttttggc
//
