LOCUS       sequence-227212-        7053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MrRT-Cas1-Cas2 under pT7lac promoter, encodes MrCRISPR 
            array 3, compatible with SENECA acquisition readout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7053)
  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 7053)
  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_0361_pET30-RT-CRISPR_Micromonospora_rosaria_ARRAY_3
FEATURES             Location/Qualifiers
     source          1..7053
                     /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(6966..6984)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6980..7027
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ccaggtgcca cccccggtgc ttgttggcca gcttgtcccc ggggcgatga gtgactggtc
       61 ggcgagggtg ggcagcccag tgtcgtacga gtttcgatcc cagggattgt gagcgacccg
      121 agcgcggcac gtgggccgca tacgtgacgg cgactcgttg cgatccctcc aggggtgatg
      181 agcgacgtcc 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 tacatatgac
     4921 cagggtgtct atccctaaac ccgatggcgg aatacgtagc ttggctatag gagctattga
     4981 agatagaatt gtcgaaagag ctgttctgga tgttcttgat ccagttgtgg accccacact
     5041 ttccccgtgg agttttgcat acaggagagg cttaggcgtg agggatgcag ttcgagcttt
     5101 ggcagaagct agggaatctg gtctagcttt tgttgttcgt tgtgatattg atgattgttt
     5161 tgattcgatc ccgagatggc ctctacttag gagactaaga gaattggttt ctgacgctga
     5221 attggttgcc ttggtagaaa gattggttgg cagaccagtc actggagaaa gggcttccgg
     5281 tggtagggga ttgcatcagg gcggatcgct cagtcctttg ttggccaacc tatatttgga
     5341 tacctttgat cgagcattga tgcgtcatgg acaccgtgta gttagatatg gagatgatat
     5401 agctatatct gttcccgata ggccaactgg acttagagta ttggatttgg ctgatgcaga
     5461 agcagaagcc ctgtctttga gactaaatac tgatgataga caagtcatag cttttgatga
     5521 aggagtccct ttctgtggtc aggttgttac tgcatcaagc gggccaacag ctgatttaca
     5581 agctaaacct ctgcaaggta cagtctttgt tacaacccaa ggagcattgc taagagtaaa
     5641 aggagaacga cttcgtgtcg aagatggaga caggctactg gctaatgtaa atttgaagcg
     5701 tgtgaggcaa atagtctgtt ttggtagagt aggagtgact tctaccttgt tacaaagaat
     5761 tgtagaacga ggaatagaac ttgcttggtt atatgaggat ggtagacatg cagctagagt
     5821 aagtgggttg gatggtactg atccagaggt ccgtttagcc caatatagag ctgctgatga
     5881 tgcacgtcag gcactgagaa tagctaggca gttagtcgct ggtaaagtaa caaatatgag
     5941 agttggtctt ttgcgagctg ctagggctca acaagctcca gagcttgctg atcgtcaggc
     6001 acgattggcc acagctcgac agtcagcttt gattgcagat tctactgctg aattgatggg
     6061 ttatgagggt tcagctacta gagattattt tgctggtctt tcacaaattt tgggtcctga
     6121 gtggggcttt actactcgtc aaagaaggcc tcctcctgac cccgtgaatg ccatgcttag
     6181 ttttggatat accttgctga ctaatgaggc actcacggcc tgtcagctgg ctggacttga
     6241 tccatatctc ggtatgttac attctcctag aagaaataga ccttctttgg cattggattt
     6301 aatcgaagaa ctccgacccg ttgttgttga tgctaccgtt attaggctag tacgaacagg
     6361 acaagttact cctaagaact ttactctgac agatgataga ggatgtagat tggatgatca
     6421 tggtagacgt gcctttctgg atgcttatga gcgtagaatg ctaacacttg tccatcatcc
     6481 tgtagaacaa agaagaatac cgtggagaca tgttttgtta gcacaagcta gaacacttgc
     6541 tgctgtattg tcatcgcgtc gtcccgaata tagacccgta gtatggcgtt aacgtacgtt
     6601 aactttaaga aggagaactt aagatgtccc gtgtttgtta tgtagttgcc tatgatatag
     6661 ccgatgacga ccgaagggcc gatctcgcta tgtttctttc agggtacggt ccgcgtgttc
     6721 agttgtctgt gtttgaagtc gaattgccgg atgctgatac agctgttgct tttagggagc
     6781 gtttgcgtag tttaatagac cctaacgacg atcaagttcg tctgtatcga ctcactccac
     6841 aggcattggg acagagaatt atttacggaa gaaggaccat tgaggagaga gttgactttt
     6901 ggatcgtgta actcgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     6961 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     7021 ttttttgctg aaaggaggaa ctatatccgg ata
//
