LOCUS       sequence-226671-        7050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressed KpAGO 207-1251 in E. coli under IPTG-inducible promotor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7050)
  AUTHORS   Nakanishi K, Weinberg DE, Bartel DP, Patel DJ
  TITLE     Structure of yeast Argonaute with guide RNA.
  JOURNAL   Nature. 2012 Jun 20;486(7403):368-74. doi: 10.1038/nature11211.
  PUBMED    22722195
REFERENCE   2  (bases 1 to 7050)
  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"; journalName: 
            "Nature"; date: "2012-06-20- 20"; volume: "486"; issue: "7403"; 
            pages: "368-74"
COMMENT     Sequence Label: pRSF-KpAGO207-1251
FEATURES             Location/Qualifiers
     source          1..7050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     164..183
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(203..280)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     326..345
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        326..344
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    345..369
                     /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)."
     CDS             425..442
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             452..469
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             482..775
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3929..3973
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4011..4029)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4025..4072
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4302..5117)
                     /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(4302..5117)
                     /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     5025..5044
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5118..5209)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5225..5974)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(5235..5252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5475..5494)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6170..7050,1..202))
                     /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"
ORIGIN
        1 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
       61 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
      121 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
      181 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
      241 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
      301 ttatgcgact cctgcattag gaaattaata cgactcacta taggggaatt gtgagcggat
      361 aacaattccc ctgtagaaat aattttgttt aactttaata aggagatata ccatgggcag
      421 cagccatcat catcatcatc acagcagcgg cctggtgccg cgcggcagcc acatggcaag
      481 catgtcggac tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc
      541 tgagactcac atcaatttaa aggtgtccga tggatcttca gagatcttct tcaagatcaa
      601 aaagaccact cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat
      661 ggactcctta agattcttgt acgacggtat tagaatccaa gctgatcaga cccctgaaga
      721 tttggacatg gaggataacg atattattga ggctcacaga gaacagattg gtggatccac
      781 cgagaaaact gtagatcaac ctaaggaaga tactcatgca atctataaag ttgaaaatag
      841 acatgattat ggtactaaag gtactaaagt tgacattttg acaaaccata ttttgcttgc
      901 ggtaggtaac gatgttccta ctgaaaaaat cgacaaagaa ttggttccta aattagatgg
      961 ttggtggaaa actgccttca tctttactta tcatatcgat ttcaaacccc aacaaaaagg
     1021 ccctccacgt agaggcaaac cagttccacc acaggaatta tcaaagccaa agaaatatga
     1081 attaattgaa gctttactgg atgaagatga aatcttgtac aagtataggg atcgtatcgc
     1141 tttcaatggt gaagatacta tttattctca tgtgccacta gaagaattca ctttgtttga
     1201 tggttgttgg gaagtcagta acaagcaaaa gaagaaggtt gttccaggta tgggtgctcc
     1261 atccaacaaa gcttctctac aaaaaaaaat agatccagaa ttagaagaaa tggtttctca
     1321 aatcactttg aaattcagtg gtaaagtagg cttgaaagat atttataacg acaccactac
     1381 tcaagacacc gaagtacagg aaagtagaat gtctgcaatc gataagacat gtttgctatc
     1441 tttattaggt gctaagttca tgagtacaga tgatttgatt ttccaagttc aaggtaacaa
     1501 gttctttatt ttcaataatt ttgctaaagc tatcccattc caaatcggtg gttatttgtt
     1561 gcagggtttt actgtttcat taactcatgt ttatggtggt gtcgctttaa acactgtcag
     1621 tgttcctgct ccatttatta aacataccaa gtacttgcca ggtgatccaa gatttaaaaa
     1681 caatgaaaag gaacaattta cattaatgga ctggatcatt gaatgttacc accaatctaa
     1741 ggctataaga gatatcagat ataatccaaa aacagctcct ccaccatcag ttaaagattt
     1801 gaactatttt gtcgaaaaga acactgatat ttcagctttg ttaaagggtt taaaggttta
     1861 cagaccgtac atcaattata gtatcaataa agatggtacc ccaaaaccac caagaaagag
     1921 atcttctaag ggtattgttg gatttacccg tgaatctgcg gtatccatga ggtttaatgt
     1981 tcttgaaagt agtttgaaga agaacagtgc tccaaaacct aatgaaaaac ctatcaatat
     2041 caatactatt gattatttca agaggaaata tgacattact ttgaaatatc ctgatatgaa
     2101 gttagtaaac ttgggcggta aaaatgatgt cgttcctcct gaatgtttga ctattgtgcc
     2161 gggtcaaaaa ttgaagggtc aaatttttga tacaaaaact tatatcgact tcagtgcaat
     2221 tagaccaact gaaaagtttg atttaatctc caggttatct atgccagcta taaaaagagg
     2281 gttaactgat tctgaaaagg aagaatcatc agctcctcac aatagtgctt atcaatttat
     2341 gagagtacca tctcgtattc tagatgcccc agttgttcag ttcaaagaat ctacctttga
     2401 atataaggac aagagttatg gaactaagca tgaagaatct aaaggtaact ggaacatgaa
     2461 aggtcaccaa tttatttcca ctccagccaa acaggtcaac ttaagagcaa tatttattaa
     2521 taatgctaac acagctccac cagcatctat ggaaagtgaa cttgacatct ctatggataa
     2581 attcgcatct gatgttaaac aattaggtgt ggacttcaac gtatcaggta aaccaattct
     2641 aattaatcaa tttggtcccc caattaagaa attccaaggt ggtggccgtg gtggccgtgg
     2701 tggtcgtggt agccgtggtg gccgtggtgg ccgtggtgct ccatctggtc ctccaacttt
     2761 cgaaacctct ccaggtgaga tatctttgtt aaacttatta gaaaatattc caagcaatac
     2821 ctatattttg tatgtattgc gccgtggtaa cgattctgct gtttatgata gattgaaata
     2881 tatcactgat ttgaaatttg gtgcattgaa ttcctgtgtt gtttgggaca acttcaaaaa
     2941 gaattctatt caatataatt ccaatgttgt tatgaagatg aacttgaagt tattaggtag
     3001 taaccactct ctatctattg aaaacaacaa actattaatt gataaggaat ctaacttgcc
     3061 aatattagtg ttgggttctg atgtgacaca ttatcctgaa aaggatcaaa actctattgc
     3121 ctcgttagta ggttcatacg atgacaaatt tacccaattc cctggtgatt acatgcttca
     3181 agatggtcca ggtgaagaaa taattactaa tgtcggttca ttaatgttga acagattaaa
     3241 gatatatcaa aaacataata atggtaaatt accaacgaag atcatgtact tcagagatgg
     3301 tgtttcagtt gaccaattct ctcaagttgt taagattgaa gttaagtcta ttaaggaatc
     3361 agtccgtaaa tttggtcctc aattaaatgg tggtaacaaa tacgatccac cagttacatg
     3421 tattgccact gtcaaaagaa atcaagtcag atttattcct atccaagaaa atgcgaagaa
     3481 tgaaaagggt gaagaagttg ctgttcaatc catgggtaat gttatgccag gtactgttgt
     3541 agaccgtggt atcacatctg tggcacactt tgatttcttt atccaatctc atcaagcttt
     3601 gaagggtact ggtgtcccat gccactattg gtgtctatat gacgaaaacc aatctacttc
     3661 tgactactta caagaaatat gtaacaactt atgttacatt ttcggtagat ctactaccag
     3721 tgtaaaagtc ccagccccag tatattatgc cgatttgttg tgtacccgtg ctacatgctt
     3781 tttcaaggca ggttttgaac ttaatatggc tcaagcacca aaggagaagg gttctaagga
     3841 tcaacctact gtttccaaga atgttctatt accacaagtt aacgataaca ttaaatccgt
     3901 aatgtattac atttgactcg agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca
     3961 gcacatggac tcgtctacta gcgcagctta attaacctag gctgctgcca ccgctgagca
     4021 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc
     4081 tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca
     4141 gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc
     4201 gggtctccgc aagtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4261 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     4321 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     4381 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     4441 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     4501 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     4561 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     4621 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     4681 acgcggtcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     4741 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     4801 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     4861 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     4921 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     4981 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     5041 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     5101 cgttgaatat ggctcatact cttccttttt caatattatt gaagcattta tcagggttat
     5161 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggcatgcag
     5221 cgctcttccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg
     5281 gtgtcagctc actcaaaagc ggtaatacgg ttatccacag aatcagggga taaagccgga
     5341 aagaacatgt gagcaaaaag caaagcaccg gaagaagcca acgccgcagg cgtttttcca
     5401 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagccaga ggtggcgaaa
     5461 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5521 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5581 gctttctcat agctcacgct gttggtatct cagttcggtg taggtcgttc gctccaagct
     5641 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5701 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ttggtaactg
     5761 atttagagga ctttgtcttg aagttatgca cctgttaagg ctaaactgaa agaacagatt
     5821 ttggtgagtg cggtcctcca acccacttac cttggttcaa agagttggta gctcagcgaa
     5881 ccttgagaaa accaccgttg gtagcggtgg tttttcttta tttatgagat gatgaatcaa
     5941 tcggtctatc aagtcaacga acagctattc cgttactcta gatttcagtg caatttatct
     6001 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt
     6061 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     6121 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     6181 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     6241 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     6301 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     6361 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     6421 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     6481 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     6541 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     6601 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     6661 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     6721 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     6781 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     6841 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     6901 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     6961 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     7021 gcgacggcgc gtgcagggcc agactggagg
//
