LOCUS       sequence-226996-        7269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EsRT-Cas1-Cas2 under pT7lac promoter, encodes EsCRISPR 
            array 2 RC, compatible with SENECA acquisition readout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7269)
  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 7269)
  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_0392_pET30-RT-CRISPR_Eubacterium_saburreum_DSM_3986_ARRAY_2_RC
FEATURES             Location/Qualifiers
     source          1..7269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(403..1218)
                     /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(403..1218)
                     /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     1126..1145
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1210..1229
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1340..1928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1829..1848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2082..2099
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2114..2253
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2179..2198)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2339..2361
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2355..2546)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3355..4437)
                     /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     4399..4418
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4438..4515)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4721..4740
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4828..4847
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4828..4846
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4847..4871
                     /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             4886..4908
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(7182..7200)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7196..7243
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ccaggtgaac actgaacgtt gctcatgtgt aaggtatttt cggatggttt tcatcacctt
       61 gggatctcgc ttagccttaa catcatatgt gataatgacg aacatctctt cctccttgta
      121 ttttgatttt agtattccaa cctggtgaat ataggtgttt ccattcctta taggtaaggt
      181 acttacgtcc cgcggccgct attcttttga tttataaggg attttgccga tttcggccta
      241 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
      301 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
      361 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
      421 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
      481 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
      541 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
      601 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
      661 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
      721 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
      781 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
      841 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
      901 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
      961 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     1021 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     1081 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     1141 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     1201 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     1261 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1321 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1381 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1441 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1501 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1561 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1621 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1681 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1741 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1801 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1861 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1921 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggtcttttgc tggccttttg
     1981 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     2041 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     2101 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2161 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     2221 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     2281 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     2341 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     2401 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     2461 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     2521 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     2581 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     2641 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     2701 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     2761 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     2821 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga tcattcatgt tgttgctcag
     2881 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     2941 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     3001 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     3061 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     3121 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     3181 gctgccggca cctgtcctac gagttgcatg ataaagaaga tagtcataag tgcggcgacg
     3241 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     3301 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     3361 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3421 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     3481 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     3541 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     3601 gctatcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga
     3661 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     3721 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     3781 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     3841 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     3901 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccatcttcat gggagaaaat
     3961 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     4021 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     4081 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     4141 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     4201 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     4261 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     4321 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     4381 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     4441 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     4501 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     4561 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     4621 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     4681 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     4741 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     4801 tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     4861 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgaa
     4921 gaaactgact atggaagata tcttcatatc cgagcaaatc gaggaagccc tcgatcacct
     4981 gtctactaag aaggacacat gcggtattga cggtttgtac ttgtccgagc ttagggacga
     5041 ctggaacatc aacggtgaga ggtacttgtc cctcttgcgt aaggggaaat ataagccggg
     5101 tatcgtgcag atttacgaga ttgttaacta caccgggaag cgtagatcca tttcctcttt
     5161 taattccata gataggctag tcctgagatg ccttgctaca agtctggaga agtactacga
     5221 ctcgatcttc tctagctcct cgttcgcttt ccgaccaggg ctcggggtgg acaaagcagt
     5281 cgccacgttc gccaacaacc tgaacaccgg actgaccagg gtagcaatca tcgacatcaa
     5341 acactacttc gattctattc caatcgatcg tttggagatg atactgaagc gaatcataga
     5401 cgacaacgtc ttattgagtc tattccacaa cttactttat tgccgtatct ctgaggagaa
     5461 cgtaatcaag actaagtcta agggcatact ccagggatca ccaatatctc cattccttgg
     5521 taacttgtac ttatctttac tagatacaca gctggagtct atgcacgtat cattttgcag
     5581 atactgcgac gacatcgcta tgttcttcgc ttctttcgag gaagctaagg aaacttacac
     5641 taaggtctac gatatcttga agaacgacct ggagatggac attaacccac agaagtcagg
     5701 gatctacgaa ggcatcaagc agaactactt gggttatagt ttcactaaga acaagaagga
     5761 gcaccagatc ttagctatca agaagaagaa ggctccacct cagatttacc agcactggtc
     5821 caccaccgca atccagaggg tcgataggaa ttatcatatc attaacaacg gtattcttaa
     5881 ccgtaaggac ttcaccctct tgttcgagaa cgacagaggc aagaaatatc tccctgttga
     5941 agccaccgag tccctaaacg tgtacacaag tgtaattttc tcctcagact tcttcaagta
     6001 cgtaggcaac aagaaaatct gcgtcaacat ctttgataag tatggggagt tggccggaac
     6061 tttcagtcca ccggagtccc ttcacggtgg cctcaccatg ttaaaacaag ctgccattta
     6121 cttggacgac gagaaaagga agctgatcgc caggaaattg gaaattgcat ccctgcataa
     6181 catgaggtct aatctgaagt actatgagcg tcaccactcc tctgaaaacc tgaaggacgg
     6241 tattacctcg ttctctgaat acattaccgc tatgaacgag gctacaaaca tcgttatgct
     6301 attaaccatt gaggcacgag cacgtcagtt gtattattct ctgtttcacg agatcatttg
     6361 cgacccagct ttcgagttca cgaagcgtac ccgtaggcca ccaaaagacc ccttgaatgc
     6421 cttaatctca ttcggcaacg ttttcttgta taacagaatc gccacggaaa tccagaaaac
     6481 atcgttggac ataaggatcg ggttcgttca cgctacaaac aggcgtaacc aaagcctgaa
     6541 cctagatata gccgacctat tcaagcccct gatcgtggat agagctattt tcaccatcat
     6601 taaccgacac atgatccacg cttcagagca cttcgagaaa acagaggacg gtggcattta
     6661 cctcaacaaa gagggtaagc agatcttcat taacgagttg gagaacaagg tttatcagaa
     6721 acaaacagag gagaacaaac ctcgtaccta cgatactcgt atccgagagg agatccacaa
     6781 gattttccga ttagtttgtt acgacgagaa gtataagcca ttcaagtata actaacgtac
     6841 gttaacttta agaaggagaa cttaagatgt ttgtaatcat aacctacgac gtaaaggcca
     6901 agagggaccc gaaggtcatg aaaactataa ggaagtactt aacccacgag cagaggtctg
     6961 ttttcgaggg gctcatcacg cccggtagat tgaagcactt gaaggaagaa cttaaaagga
     7021 ttgtgaacgt gtcagaggac tgtatcaaca tttactccct ggaaaccctg cgttacagta
     7081 agaaggagtc catcgggaag caagtttacc acggcaacat catttaactc gagatccggc
     7141 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     7201 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     7261 atccggata
//
