LOCUS       sequence-473288-        6649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TaTasR with an N-terminal Twin-Strep-SUMO tag. KanR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6649)
  AUTHORS   Faure G, Saito M, Wilkinson ME, Quinones-Olvera N, Xu P, 
            Flam-Shepherd D, Kim S, Reddy N, Zhu S, Evgeniou L, Koonin EV, 
            Macrae RK, Zhang F
  TITLE     TIGR-Tas: A family of modular RNA-guided DNA-targeting systems in 
            prokaryotes and their viruses.
  JOURNAL   Science. 2025 Feb 27:eadv9789. doi: 10.1126/science.adv9789.
  PUBMED    40014690
REFERENCE   2  (bases 1 to 6649)
  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. 
            2025 Feb 27:eadv9789. doi: 10.1126/science.adv9789."
COMMENT     Sequence Label: pMIIS793
FEATURES             Location/Qualifiers
     source          1..6649
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(560..1375)
                     /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(560..1375)
                     /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     1283..1302
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1367..1386
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1986..2005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2239..2256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2271..2410
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2336..2355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2496..2518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2512..2703)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3512..4594)
                     /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     4556..4575
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4595..4672)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4878..4897
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4981..5000
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4981..4999
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5000..5024
                     /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             5039..5061
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5080..5097
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5107..5124
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(5137..5160,5161..5196,5197..5220)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             5221..5514
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(6563..6581)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6577..6624
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaact agtaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatgt
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     2341 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     2401 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     2461 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     2521 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     2581 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     2641 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     2701 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     2761 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     2821 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     2881 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     2941 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     3001 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagaagca
     3061 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     3121 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     3181 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     3241 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     3301 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     3361 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     3421 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     3481 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3541 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3601 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     3661 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     3721 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     3781 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     3841 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgataccc tcattcagca
     3901 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     3961 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     4021 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     4081 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     4141 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     4201 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     4261 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     4321 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     4381 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     4441 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     4501 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     4561 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     4621 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     4681 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     4741 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     4801 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     4861 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     4921 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     4981 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     5041 tgtttaactt taagaaggag atatagcatg ggcagcagcc atcatcatca tcatcacagc
     5101 agcggcctgg tgccgcgcgg cagccatatg gctagctgga gccatccgca gtttgaaaaa
     5161 ggtggtggta gcggtggtgg ttcaggtggt agtgcatggt cacaccctca gtttgagaaa
     5221 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
     5281 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa
     5341 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
     5401 gactccttaa gattcttgta cgacggtatt agaatccaag ctgatcagac ccctgaagat
     5461 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggatccgat
     5521 aacgatatca caatattggc ggtcgattgg tctcatgaag aacgtaaact tgcaattttt
     5581 gatggcaaga agataaggaa aaaacttcct gagccttcta gcgatgtaat aattgtggct
     5641 gaaaatattc ctcaaaaata tgccgctccg ttcatcgagg taggggcaaa agttcttaga
     5701 tgttcgacaa acgccacagc agatgcaaga aaaaactatc aaaagaaagt cgatgctgct
     5761 tttgcgaaaa acgacgaaaa tgattctaaa gtgatttggg cgttatatca aacacaccca
     5821 gaattgtttc gtgaaatgaa gcttgagcca cccctttctt cttattatgc tatttttaaa
     5881 gattaccaag aggtaagaat tagaactggc aatcgactct attcagatcg cactgatgca
     5941 atggaagagt tttttaaaat agttaagaaa ggtgaacatg aactaaaaaa ggctgtcgat
     6001 aaagaattag aaaatcatcc ggtctatacg cagtggttac agcatattaa aggtattggc
     6061 ccggtcgttg ccgggggatt gatatcgtta attggagata tagatcgctt cgacagcgtt
     6121 tctaaattat gggcctatgc aggttatagt gttgataatg gaaaagtgca aaagcgtaaa
     6181 aaaggtgtag cttcaaattg gaaaaataaa attcgcacac attgttacaa cattgtagat
     6241 tcatttatca aacaacgcac ttcagtttat cgtgagttgt atgatgccga aaaagctcgt
     6301 caacgtccaa aggtagaatc cgatggtcat gctcacaacc gtgctgtaag aaaagttgct
     6361 aaggtatttt tacaacatta ttgggtagtt tctagagaat tagcagggtt ttctgtgtct
     6421 aaaccttgga ttttagaaca tggtgggcac gttgattaca tcaaaccacc acattggaat
     6481 aaagtagaaa ttaagccatg caagccctga tgagatccgg ctgctaacaa agcccgaaag
     6541 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     6601 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat
//
