LOCUS       sequence-417961-        7701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the His- and SUMO-tagged catalytically 
            inactive Argonaute protein of Thermus thermophilus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7701)
  AUTHORS   Jolly SM, Gainetdinov I, Jouravleva K, Zhang H, Strittmatter L, 
            Bailey SM, Hendricks GM, Dhabaria A, Ueberheide B, Zamore PD
  TITLE     Thermus thermophilus Argonaute Functions in the Completion of DNA 
            Replication.
  JOURNAL   Cell. 2020 Sep 17;182(6):1545-1559.e18. doi: 
            10.1016/j.cell.2020.07.036. Epub 2020 Aug 25.
  PUBMED    32846159
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"; doi: 
            "10.1016/j.cell.2020.07.036"; journalName: "Cell"; date: 
            "2020-09-17- 17"; volume: "182"; issue: "6"; pages: "1545-1559.e18"
COMMENT     Sequence Label: pETSUMO-TtAgocatdead
FEATURES             Location/Qualifiers
     source          1..7701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     459..478
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     712..729
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    744..883
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(809..828)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     969..991
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(985..1176)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1985..3067)
                     /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     3029..3048
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3068..3145)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3351..3370
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3454..3473
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3454..3472
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3473..3497
                     /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             3512..3534
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3554..3571
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3581..3598
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3605..3898
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             6019..6036
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6089..6107)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6103..6150
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6187..6642
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6274..6293)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6484..6505
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6734..7549)
                     /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(6734..7549)
                     /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     7457..7476
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7541..7560
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(7671..7701,1..558)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
       61 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
      121 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      181 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      241 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      301 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
      361 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      421 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      481 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      541 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
      601 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
      661 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
      721 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
      781 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
      841 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
      901 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
      961 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     1021 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     1081 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     1141 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     1201 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     1261 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     1321 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     1381 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     1441 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     1501 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     1561 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     1621 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     1681 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     1741 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     1801 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     1861 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     1921 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     1981 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2041 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     2101 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     2161 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     2221 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     2281 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     2341 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     2401 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     2461 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     2521 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     2581 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     2641 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     2701 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     2761 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     2821 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     2881 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     2941 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     3001 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     3061 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     3121 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     3181 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     3241 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     3301 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     3361 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     3421 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     3481 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca
     3541 tatgggcagc agccatcatc atcatcatca cggcagcggc ctggtgccgc gcggcagcgc
     3601 tagcatgtcg gactcagaag tcaatcaaga agctaagcca gaggtcaagc cagaagtcaa
     3661 gcctgagact cacatcaatt taaaggtgtc cgatggatct tcagagatct tcttcaagat
     3721 caaaaagacc actcctttaa gaaggctgat ggaagcgttc gctaaaagac agggtaagga
     3781 aatggactcc ttaagattct tgtacgacgg tattagaatt caagctgatc agacccctga
     3841 agatttggac atggaggata acgatattat tgaggctcac agagaacaga ttggtggtat
     3901 gaaccacctt ggaaaaacgg aagtgttcct aaaccgcttc gccctacggc ctctgaaccc
     3961 cgaggaactc aggccttggc gcctcgaggt ggttctggac cctcctccgg ggcgggagga
     4021 ggtgtatccc cttctcgccc aggtggcccg ccgggcgggg ggcgtcacgg tgcgcatggg
     4081 agacggcctc gcctcctggt caccccctga ggtcctggtc ttggagggca ccttggcgcg
     4141 gatggggcag aactacgcct accgcctcta ccccaagggg aggaggcctc tggaccccaa
     4201 ggacccgggg gagcggagcg tgctttcggc cctagcccga aggctccttc aggagcgcct
     4261 caggcgcctc gagggggtct gggtggaggg gcttgcggtg taccggaggg agcacgcccg
     4321 ggggcccggg tggcgggtgc ttgggggggc ggtcttggac ctttgggtct ccgactcggg
     4381 ggcgttcctc ctggaggtgg accccgctta ccgcatcctc tgcgagatgt ccctcgaggc
     4441 ctggcttgcc caaggccacc ctctgcccaa acgggtccgg aacgcctacg accggcgcac
     4501 ctgggagctt ctccggctgg gagaggaaga ccccaaggag ctccctctcc cgggcggctt
     4561 gagcctcctg gactaccacg ctttcaaggg ccgcctccag ggccgggaag gggggcgggt
     4621 ggcctgggtg gcggacccaa aggatccgcg aaagccaatc ccccatctga cgggcctttt
     4681 ggtgcccgtt ctgaccctgg aagaccttca tgaagaggag ggaagcctgg ccctctcctt
     4741 gccttgggaa gagcggcgtc ggcggacccg ggagatcgcc agctggatcg gccggcgcct
     4801 ggggctgggc acacctgagg cggtgcgcgc ccaggcttac cgcttgagca tccccaagct
     4861 catgggcaga agagccgtga gcaagcctgc ggacgccctc cgcgtggggt tttaccgggc
     4921 ccaggaaacc gccctggccc tcttgcgcct ggacggggcc caaggatggc cggagtttct
     4981 ccggcgggcc ttgctccggg cctttggggc gagcggggct tccctccgtt tgcacaccct
     5041 ccacgcccat ccctcccaag gcctggcttt ccgcgaggcg ctgcggaagg ccaaggagga
     5101 gggggtccag gccgtgctgg tcctcacccc gcccatggcc tgggaagacc gcaaccgcct
     5161 gaaggccctt cttctcaggg aaggccttcc cagccaaatc ctcaacgtcc ccctccggga
     5221 ggaggaacgc caccgctggg agaacgccct cctgggcctc ctggccaaag cggggcttca
     5281 ggtggtggcc ctgagcggcg cctatccggc ggagctcgcc gtgggctttg ccgccggcgg
     5341 aagggagtcc tttcgcttcg ggggcgcggc ctgcgccgtg ggcggggacg gcggccacct
     5401 cctctggacc ctccccgagg cccaggccgg ggagcggatc ccccaggagg tggtctggga
     5461 cctcttggag gagaccctct gggccttcag acgcaaggcg gggaggcttc cttcccgggt
     5521 cctcctcctt cgggccggcc gcgtgcccca ggacgagttc gccctggcct tggaggccct
     5581 tgcccgggaa ggcatagcct acgacttggt ttcggtgcgc aagtcgggtg gggggcgggt
     5641 ctaccccgtg caggggcgcc tggcggacgg gctttacgtc cccttggagg acaagacctt
     5701 tcttctcctt accgtccacc gggacttccg gggcacgccc cgacccctga agctggtgca
     5761 cgaggcgggg gacacgcccc tcgaggccct ggcccaccag attttccatc tgacccgcct
     5821 ctacccggcg agcggtttcg ccttcccccg gcttcccgct ccccttcacc tggccgaccg
     5881 cctggtgaag gaggtgggcc ggttggggat ccgtcacctc aaggaggtgg accgggaaaa
     5941 gctcttcttc gtttagagac aagcttaggt atttattcgg cgcaaagtgc gtcgggtgat
     6001 gctgccaact tagtcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     6061 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     6121 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     6181 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     6241 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     6301 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     6361 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     6421 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     6481 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     6541 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     6601 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     6661 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     6721 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     6781 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     6841 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     6901 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     6961 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     7021 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     7081 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     7141 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     7201 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     7261 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     7321 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     7381 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     7441 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     7501 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     7561 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     7621 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7681 ttttttctgc gcgtaatctg c
//
