LOCUS       sequence-394510-        8232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial protein expression of Dhh1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8232)
  AUTHORS   Currie SL, Xing W, Muhlrad D, Decker CJ, Parker R, Rosen MK
  TITLE     Quantitative reconstitution of yeast RNA processing bodies.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Apr 4;120(14):e2214064120. doi: 
            10.1073/pnas.2214064120. Epub 2023 Mar 27.
  PUBMED    36972455
REFERENCE   2  (bases 1 to 8232)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-04-4"; volume: "120(14):e2214064120. 
            doi"; pages: " 10.1073/pnas.2214064120. Epub 2023 Mar 27"
COMMENT     Sequence Label: Dhh1
FEATURES             Location/Qualifiers
     source          1..8232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(94..99,100..115,116..122)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    130..146
                     /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)."
     primer_bind     135..157
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             216..1316
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1173..1182
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1290..1313
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1365..1376
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1389..1409
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2937..2957
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2958..2975
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2988..3005)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2988..3004)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2997..3019)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3183..3200)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3353..3439
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3531..3558
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3577..3668
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3669..3737,3738..4529)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3887..3906)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4571..5084)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4766..4787)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4978..4997
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5195..5783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5684..5703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5937..5954
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(6060..6079)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6220..6242
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6236..6427)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6923..7000
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             7001..8083
                     /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     complement(7020..7039)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
       61 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa
      121 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagccagtcc gtttaggtgt
      181 tttcacgagc acttcaccaa caaggaccat agattatgaa aactgaagaa ggtaaactgg
      241 taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg
      301 agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc
      361 cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg
      421 gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca
      481 agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga
      541 tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa
      601 cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga
      661 tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg
      721 cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg
      781 cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca
      841 ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg
      901 gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc
      961 cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg
     1021 ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg
     1081 aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg
     1141 aggaagagtt ggcgaaagat ccacgtattg ccgccaccat ggaaaacgcc cagaaaggtg
     1201 aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga
     1261 tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt
     1321 cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag ggaaggattt
     1381 cagaatttga aaacctgtat tttcagggcc atatgggttc catcaataat aacttcaaca
     1441 ctaataataa cagtaatacg gatctcgatc gggactggaa aactgcattg aatattccca
     1501 agaaggatac cagaccacag acagatgatg tcttaaatac aaagggtaat acttttgaag
     1561 atttttattt gaaaagagag ctgttaatgg gtatttttga agccggtttt gaaaaaccat
     1621 cccctattca agaagaggct attccggtag caataactgg tagagacata ttggccagag
     1681 caaagaatgg tacaggtaag acagccgcat ttgttattcc tacgttagag aaagtcaaac
     1741 caaagttgaa taaaattcag gctttgatca tggttcccac aagagagtta gctctgcaaa
     1801 cttcccaagt cgtccgtaca ttgggaaaac actgtggtat ttcatgtatg gtgaccactg
     1861 gtggtaccaa tttgagagac gatattctaa gattaaatga aacagtccat attttggttg
     1921 gtactcctgg tagagtgttg gatttagctt cgagaaaagt agcagatctg tctgattgtt
     1981 cattattcat catggacgaa gccgataaaa tgttatctcg tgacttcaag acaataattg
     2041 aacaaatctt atccttctta cctccaactc accaatcttt attatttagc gctactttcc
     2101 cactaacggt taaggaattc atggttaaac atctgcataa gccttatgaa attaatttga
     2161 tggaagaatt gactctaaag ggtattactc aatactacgc ctttgttgaa gaaaggcaaa
     2221 agctacattg tttaaatact ttattctcta agcttcaaat taatcaagcc attatttttt
     2281 gtaattctac taaccgtgtc gaattactag ccaagaaaat cactgattta ggctattcct
     2341 gttattactc tcatgcaaga atgaagcaac aagaaagaaa taaagttttc cacgaatttc
     2401 gtcaaggtaa ggttcgtaca ttggtctgct ccgatttatt gacccgtggt atcgatattc
     2461 aagccgttaa tgtcgttatc aatttcgatt tccccaaaac ggcagaaaca tatttacatc
     2521 gtattggtag atccggtagg tttggccact tgggtttagc aatcaatttg attaactgga
     2581 atgatcggtt taacctttat aaaatcgaac aagaactggg cactgaaatt gcggctattc
     2641 cggccactat cgataagtca ttgtatgtgg ctgaaaatga cgaaacggta cctgtcccat
     2701 tcccgataga gcaacagagc tatcatcaac aagcgatacc tcagcaacaa ttaccatctc
     2761 agcaacaatt tgctattcct ccgcagcaac atcatccaca attcatggtt cctccttcgc
     2821 atcagcaaca acaggcgtat cctccaccgc aaatgccatc gcagcagggg tatcctccac
     2881 agcaggaaca tttcatggcg atgccacctg gtcagtcaca accccagtat ggatccgaaa
     2941 acctgtattt tcagggccat catcatcatc atcactgaaa gcttggcact ggccgtcgtt
     3001 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     3061 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     3121 ttgcgcagcc tgaatggcga atggcagctt ggctgttttg gcggatgaga taagattttc
     3181 agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc
     3241 ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc
     3301 gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa
     3361 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     3421 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg
     3481 agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat
     3541 cctgacggat ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt
     3601 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3661 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3721 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3781 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3841 ttcgccccga agaacgtttc ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3901 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3961 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4021 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4081 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4141 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4201 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4261 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4321 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4381 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4441 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4501 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4561 agattgattt accccggttg ataatcagaa aagccccaaa aacaggaaga ttgtataagc
     4621 aaatatttaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     4681 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     4741 gcccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     4801 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     4861 atcacccaaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     4921 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     4981 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     5041 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg atctaggtga
     5101 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5161 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5221 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5281 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5341 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5401 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5461 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5521 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5581 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5641 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5701 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5761 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5821 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5881 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5941 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     6001 gcggtatttc acaccgcatg tggtgcactc tcagtacacc gcatgtggtg cactctcagt
     6061 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     6121 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     6181 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     6241 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     6301 cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     6361 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     6421 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     6481 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     6541 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     6601 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     6661 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     6721 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     6781 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     6841 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     6901 acccaacgct gcccgaaatt ccgacaccat cgaatggtgc aaaacctttc gcggtatggc
     6961 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     7021 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     7081 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     7141 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     7201 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     7261 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     7321 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     7381 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     7441 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     7501 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     7561 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     7621 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     7681 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     7741 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     7801 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     7861 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     7921 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     7981 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     8041 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     8101 taagttagct cactcattag gcacaattct catgtttgac agcttatcat cgactgcacg
     8161 gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc
     8221 gtaaatcact gc
//
