LOCUS       sequence-417962-        7701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the His- and SUMO-tagged Argonaute protein 
            of Thermus thermophilus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7701)
  AUTHORS   Smith CS, Jouravleva K, Huisman M, Jolly SM, Zamore PD, Grunwald D
  TITLE     An automated Bayesian pipeline for rapid analysis of single-molecule
            binding data.
  JOURNAL   Nat Commun. 2019 Jan 17;10(1):272. doi: 10.1038/s41467-018-08045-5.
  PUBMED    30655518
REFERENCE   2  (bases 1 to 7701)
  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: "Nat 
            Commun."; date: "2019-01-17"; volume: "10(1):272. doi"; pages: " 
            10.1038/s41467-018-08045-5"
COMMENT     Sequence Label: pETSUMO-TtAgo
FEATURES             Location/Qualifiers
     source          1..7701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     457..476
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     710..727
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    742..881
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(807..826)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     967..989
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(983..1174)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1983..3065)
                     /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     3027..3046
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3066..3143)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3349..3368
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3452..3471
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3452..3470
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3471..3495
                     /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             3510..3532
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3552..3569
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3579..3596
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3603..3896
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             6017..6034
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6087..6105)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6101..6148
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6185..6640
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6272..6291)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6482..6503
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6732..7547)
                     /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(6732..7547)
                     /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     7455..7474
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7539..7558
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(7669..7701,1..556)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
       61 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
      121 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
      181 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
      241 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
      301 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
      361 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
      421 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
      481 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      541 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
      601 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
      661 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
      721 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
      781 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
      841 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
      901 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
      961 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     1021 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     1081 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     1141 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     1201 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     1261 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     1321 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     1381 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     1441 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     1501 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     1561 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     1621 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     1681 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     1741 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     1801 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     1861 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     1921 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     1981 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2041 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     2101 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     2161 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     2221 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     2281 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     2341 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     2401 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     2461 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     2521 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     2581 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     2641 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     2701 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     2761 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     2821 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     2881 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     2941 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     3001 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     3061 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     3121 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     3181 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     3241 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     3301 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     3361 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     3421 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     3481 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
     3541 tgggcagcag ccatcatcat catcatcacg gcagcggcct ggtgccgcgc ggcagcgcta
     3601 gcatgtcgga ctcagaagtc aatcaagaag ctaagccaga ggtcaagcca gaagtcaagc
     3661 ctgagactca catcaattta aaggtgtccg atggatcttc agagatcttc ttcaagatca
     3721 aaaagaccac tcctttaaga aggctgatgg aagcgttcgc taaaagacag ggtaaggaaa
     3781 tggactcctt aagattcttg tacgacggta ttagaattca agctgatcag acccctgaag
     3841 atttggacat ggaggataac gatattattg aggctcacag agaacagatt ggtggtatga
     3901 accaccttgg aaaaacggaa gtgttcctaa accgcttcgc cctacggcct ctgaaccccg
     3961 aggagctcag gccttggcgc ctcgaggtgg ttctggaccc tcctccgggg cgggaggagg
     4021 tgtatcccct tctcgcccag gtggcccgcc gggcgggggg cgtcacggtg cgcatgggag
     4081 acggcctcgc ctcctggtcg ccccctgagg tcctggtctt ggagggcacc ttggcgcgga
     4141 tggggcagac ctacgcctac cgcctctacc ccaaggggag gaggcctctg gaccccaagg
     4201 acccggggga gcggagcgtg ctttcggcct tggcccgaag gctccttcag gagcgcctca
     4261 ggcgcctcga gggggtctgg gtggaggggc ttgcggtgta ccggagggag cacgcccggg
     4321 ggcccgggtg gcgggtgctt gggggggcgg tcttggacct ttgggtctcc gactcggggg
     4381 cgttcctcct ggaggtggac cccgcttacc ggatcctctg cgagatgtcc ctcgaggcct
     4441 ggcttgccca aggccaccct ctgcccaaac gggtccggaa cgcctacgac cggcgcacct
     4501 gggagcttct ccggctggga gaggaagacc ccaaggagct ccccctcccg ggcggcttaa
     4561 gcctcctgga ctaccacgct tccaagggcc gcctccaggg ccgggaaggg gggcgggtgg
     4621 cctgggtggc ggacccaaag gatccgcgaa agccaatccc ccatctgacg ggccttttgg
     4681 tgcccgtcct taccctggaa gaccttcatg aagaggaggg aagcctggcc ctctccttgc
     4741 cttgggaaga gcggcgtcga cggacccggg agatcgccag ctggatcggc cggcgcctgg
     4801 ggctgggcac acctgaggcg gtgcgcgccc aggcgtaccg cttgagcatc cccaagctca
     4861 tgggcagaag agccgtgagc aagcctgcgg acgccctccg cgtggggttt taccgggccc
     4921 aggaaaccgc cctggccctc ttgcgcctgg acggggccca aggatggccg gagtttctcc
     4981 ggcgggcctt gctccgggcc tttggggcga gcggggcttc cctccgtttg cacaccctcc
     5041 acgcccatcc ctcccaaggc ctggctttcc gcgaggcgct gcggaaggcc aaggaggagg
     5101 gggtccaggc cgtgctggtc ctcaccccgc ccatggcctg ggaagaccgc aaccgcctga
     5161 aggcccttct cctcagggaa ggccttccca gccaaatcct caacgtcccc ctccgggagg
     5221 aggaacgcca ccgctgggag aacgccctcc tgggcctcct ggccaaggcg gggcttcagg
     5281 tggtggccct gagcggcgcc tatccggcgg agcttgccgt gggctttgac gctggcggaa
     5341 gggagtcctt tcgcttcggg ggcgcggcct gcgccgtggg cggggacggc ggccacctcc
     5401 tctggaccct ccccgaggcc caggccgggg agcggatccc ccaggaggtg gtctgggacc
     5461 tcttggagga gaccctctgg gccttcagac gcaaggcggg gaggcttcct tcccgggtcc
     5521 tcctccttcg ggacggccgt gtgccccagg acgagttcgc cctggccttg gaggcccttg
     5581 cccgggaagg catagcctac gacttggttt cggtgcgcaa gtcgggaggg gggcgggtct
     5641 accccgtgca ggggcgcctg gcggacgggc tttacgtccc cttggaggac aagacctttc
     5701 ttctcctcac cgtccaccgg gacttccggg gcacgccccg acccctgaag ctggtgcacg
     5761 aggcggggga cacgcccctc gaggccctgg cccaccagat tttccatctg acccgcctct
     5821 acccggcgag cggtttcgcc ttcccccggc ttcccgctcc ccttcacctg gccgaccgcc
     5881 tggtgaagga ggtgggccgg ttggggatcc gtcacctcaa ggaggtggac cgggaaaagc
     5941 tcttcttcgt ttagagacaa gcttaggtat ttattcggcg caaagtgcgt cgggtgatgc
     6001 tgccaactta gtcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     6061 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     6121 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     6181 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     6241 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     6301 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6361 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     6421 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     6481 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     6541 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     6601 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     6661 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6721 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     6781 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     6841 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     6901 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     6961 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     7021 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     7081 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     7141 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     7201 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     7261 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     7321 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     7381 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     7441 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     7501 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     7561 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     7621 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7681 ttttctgcgc gtaatctgct g
//
