LOCUS       Exported                5872 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Synthetic codon optimized P. furiosus ago with N-terminal 
            Strep(II)-tag in pCDF-1b vector, expression vector for PfAgo.
ACCESSION   .
VERSION     .
KEYWORDS    pWUR790
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5872)
  AUTHORS   Swarts DC, Hegge JW, Hinojo I, Shiimori M, Ellis MA, Dumrongkulraksa
            J, Terns RM, Terns MP, van der Oost J
  TITLE     Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided 
            nuclease that targets cognate DNA.
  JOURNAL   Nucleic Acids Res. 2015 May 26;43(10):5120-9. doi: 
            10.1093/nar/gkv415. Epub 2015 Apr 29.
  PUBMED    25925567
REFERENCE   2  (bases 1 to 5872)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1093/nar/gkv415"; journalName: "Nucleic Acids Res"; date: 
            "2015-05-26- 26"; volume: "43"; issue: "10"; pages: "5120-9"
FEATURES             Location/Qualifiers
     source          1..5872
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             97..120
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             127..150
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             160..2472
                     /codon_start=1
                     /product="Argonaute protein from the archaeon Pyrococcus 
                     furiosus"
                     /label=PfAgo
                     /note="DNA-guided nuclease (Swarts et al., 2015)"
                     /translation="MKAKVVINLVKINKKIIPDKIYVYRLFNDPEEELQKEGYSIYRLA
                     YENVGIVIDPENLIIATTKELEYEGEFIPEGEISFSELRNDYQSKLVLRLLKENGIGEY
                     ELSKLLRKFRKPKTFGDYKVIPSVEMSVIKHDEDFYLVIHIIHQIQSMKTLWELVNKDP
                     KELEEFLMTHKENLMLKDIASPLKTVYKPCFEEYTKKPKLDHNQEIVKYWYNYHIERYW
                     NTPEAKLEFYRKFGQVDLKQPAILAKFASKIKKNKNYKIYLLPQLVVPTYNAEQLESDV
                     AKEILEYTKLMPEERKELLENILAEVDSDIIDKSLSEIEVEKIAQELENKIRVRDDKGN
                     SVPISQLNVQKSQLLLWTNYSRKYPVILPYEVPEKFRKIREIPMFIILDSGLLADIQNF
                     ATNEFRELVKSMYYSLAKKYNSLAKKARSTNEIGLPFLDFRGKEKVITEDLNSDKGIIE
                     VVEQVSSFMKGKELGLAFIAARNKLSSEKFEEIKRRLFNLNVISQVVNEDTLKNKRDKY
                     DRNRLDLFVRHNLLFQVLSKLGVKYYVLDYRFNYDYIIGIDVAPMKRSEGYIGGSAVMF
                     DSQGYIRKIVPIKIGEQRGESVDMNEFFKEMVDKFKEFNIKLDNKKILLLRDGRITNNE
                     EEGLKYISEMFDIEVVTMDVIKNHPVRAFANMKMYFNLGGAIYLIPHKLKQAKGTPIPI
                     KLAKKRIIKNGKVEKQSITRQDVLDIFILTRLNYGSISADMRLPAPVHYAHKFANAIRN
                     EWKIKEEFLAEGFLYFV"
     primer_bind     complement(2491..2509)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2505..2552
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2723..3514)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(3515..3606)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(4667..5749)
                     /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     5711..5730
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5750..5827)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg gcaagctggt cacatccgca gtttgaaaaa
      121 ggtgcactgg aagttctgtt tcagggtcct gggtaccaga tgaaagcaaa agttgttatt
      181 aacctggtga aaatcaacaa aaaaatcatc ccggataaaa tctacgtgta ccgcctgttt
      241 aatgatccgg aagaagaact gcagaaagaa ggctatagca tttatcgtct ggcctatgaa
      301 aatgtgggca ttgttatcga tccggaaaat ctgattattg ccaccaccaa agaactggaa
      361 tatgaaggtg aatttattcc ggaaggcgaa atcagcttta gcgaactgcg taatgattat
      421 cagagcaaac tggttctgcg tctgctgaaa gaaaatggta ttggtgaata tgaactgtcc
      481 aaactgctgc gcaaatttcg taaaccgaaa acctttggcg attacaaagt tattccgagc
      541 gttgaaatga gcgtgatcaa acacgatgaa gatttctatc tggtgatcca tatcatccat
      601 cagatccaga gcatgaaaac cctgtgggaa ctggttaata aagatccgaa agagctggaa
      661 gaatttctga tgacccataa agaaaacctg atgctgaaag atattgccag tccgctgaaa
      721 accgtgtata aaccgtgttt tgaagaatat accaaaaaac cgaaactgga ccacaaccaa
      781 gagatcgtga aatattggta taactatcac atcgagcgct attggaatac accggaagca
      841 aaactggaat tctatcgcaa atttggtcag gtggatctga aacagcctgc aattctggca
      901 aaatttgcaa gcaaaattaa aaaaaataaa aactacaaaa tctatctgct gccgcagctg
      961 gttgttccga cctataatgc agaacagctg gaaagtgatg tggccaaaga aattctggaa
     1021 tatacaaaac tgatgcctga ggaacgtaaa gagctgctgg aaaatattct ggcagaagtg
     1081 gatagcgata tcatcgataa aagcctgagc gaaatcgagg ttgaaaaaat tgcacaagaa
     1141 ctggaaaaca aaatccgcgt gcgtgatgat aaaggtaata gcgttccgat tagccagctg
     1201 aatgttcaga aaagccagct gctgctgtgg accaattatt cacgtaaata tccggttatc
     1261 ctgccgtatg aagtgccgga aaaatttcgc aaaattcgtg aaatcccgat gttcattatt
     1321 ctggatagcg gtctgctggc agatattcag aactttgcaa ccaatgaatt tcgcgagctg
     1381 gtcaaaagca tgtattatag cctggccaaa aaatacaact ccctggcaaa aaaagcacgc
     1441 agcaccaatg aaattggtct gccgtttctg gattttcgcg gtaaagaaaa agtgatcacc
     1501 gaagatctga atagcgataa aggcattatt gaagttgttg aacaggtgag cagctttatg
     1561 aaaggtaaag aactgggtct ggcatttatt gcagcacgta ataaactgag cagcgagaaa
     1621 tttgaagaaa tcaaacgccg tctgtttaac ctgaatgtta ttagccaggt ggtgaatgaa
     1681 gataccctga aaaacaaacg cgataaatat gatcgtaatc gcctggacct gtttgttcgt
     1741 cataatctgc tgttccaggt tctgagtaaa ctgggtgtta aatactatgt gctggactat
     1801 cgcttcaact acgattatat cattggcatt gatgtggcac cgatgaaacg tagcgaaggt
     1861 tatattggtg gtagcgcagt tatgtttgat agccagggtt atattcgtaa aatcgtgccg
     1921 attaaaatcg gtgaacagcg tggtgaaagc gttgatatga acgaattttt caaagaaatg
     1981 gtggacaaat tcaaagagtt caacatcaaa ctggataaca aaaaaatcct gctgctgcgt
     2041 gatggtcgca ttaccaataa tgaagaagaa ggcctgaaat atatcagcga gatgttcgat
     2101 attgaagtgg tgacaatgga tgtgatcaaa aaccatccgg ttcgtgcatt tgcaaacatg
     2161 aaaatgtatt ttaacctggg tggtgccatt tatctgattc cgcataaact gaaacaggca
     2221 aaaggcaccc cgattccgat taaactggcg aaaaaacgca ttatcaaaaa cggcaaagtg
     2281 gaaaaacaga gcattacccg tcaggatgtt ctggatatct ttattctgac ccgtctgaat
     2341 tatggtagca ttagcgcaga tatgcgtctg cctgcaccgg ttcattatgc acataaattt
     2401 gccaatgcca ttcgcaacga gtggaaaatc aaagaagaat tcctggccga aggctttctg
     2461 tattttgtgt aataacctag gctgctgcca ccgctgagca ataactagca taaccccttg
     2521 gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca
     2581 cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta
     2641 tttaacgacc ctgccctgaa ccgacgaccg ggtcatcgtg gccggatctt gcggcccctc
     2701 ggcttgaacg aattgttaga cattatttgc cgactacctt ggtgatctcg cctttcacgt
     2761 agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa
     2821 gataagcctg tctagcttca agtatgacgg gctgatactg ggccggcagg cgctccattg
     2881 cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg taccaaatgc
     2941 gggacaacgt aagcactaca tttcgctcat cgccagccca gtcgggcggc gagttccata
     3001 gcgttaaggt ttcatttagc gcctcaaata gatcctgttc aggaaccgga tcaaagagtt
     3061 cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc agcaagatag
     3121 ccagatcaat gtcgatcgtg gctggctcga agatacctgc aagaatgtca ttgcgctgcc
     3181 attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg tcgtcgtgca
     3241 caacaatggt gacttctaca gcgcggagaa tctcgctctc tccaggggaa gccgaagttt
     3301 ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa
     3361 ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag ccgtacaaat
     3421 gtacggccag caacgtcggt tcgagatggc gctcgatgac gccaactacc tctgatagtt
     3481 gagtcgatac ttcggcgatc accgcttccc tcatactctt cctttttcaa tattattgaa
     3541 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3601 aacaaatagc tagctcactc ggtcgctacg ctccgggcgt gagactgcgg cgggcgctgc
     3661 ggacacatac aaagttaccc acagattccg tggataagca ggggactaac atgtgaggca
     3721 aaacagcagg gccgcgccgg tggcgttttt ccataggctc cgccctcctg ccagagttca
     3781 cataaacaga cgcttttccg gtgcatctgt gggagccgtg aggctcaacc atgaatctga
     3841 cagtacgggc gaaacccgac aggacttaaa gatccccacc gtttccggcg ggtcgctccc
     3901 tcttgcgctc tcctgttccg accctgccgt ttaccggata cctgttccgc ctttctccct
     3961 tacgggaagt gtggcgcttt ctcatagctc acacactggt atctcggctc ggtgtaggtc
     4021 gttcgctcca agctgggctg taagcaagaa ctccccgttc agcccgactg ctgcgcctta
     4081 tccggtaact gttcacttga gtccaacccg gaaaagcacg gtaaaacgcc actggcagca
     4141 gccattggta actgggagtt cgcagaggat ttgtttagct aaacacgcgg ttgctcttga
     4201 agtgtgcgcc aaagtccggc tacactggaa ggacagattt ggttgctgtg ctctgcgaaa
     4261 gccagttacc acggttaagc agttccccaa ctgacttaac cttcgatcaa accacctccc
     4321 caggtggttt tttcgaaggt taagtcagtt ggggaactgc ttaaccttcg atcaaaccac
     4381 ctccccaggt ggttttttcg tttacagggc aaaagattac gcgcagaaaa aaaggatctc
     4441 aagaagatcc tttgatcttt tctactgaac cgctctagat ttcagtgcaa tttatctctt
     4501 caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat
     4561 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     4621 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     4681 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4741 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     4801 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     4861 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     4921 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     4981 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     5041 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     5101 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     5161 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     5221 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     5281 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     5341 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     5401 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     5461 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     5521 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     5581 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     5641 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     5701 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     5761 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     5821 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa at
//
