LOCUS       sequence-182036-        6678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6678)
  AUTHORS   Hadjivassiliou H, Rosenberg OS, Guthrie C
  TITLE     The crystal structure of S. cerevisiae Sad1, a catalytically 
            inactive deubiquitinase that is broadly required for pre-mRNA 
            splicing.
  JOURNAL   RNA. 2014 May;20(5):656-69. doi: 10.1261/rna.042838.113. Epub 2014 
            Mar 28.
  PUBMED    24681967
REFERENCE   2  (bases 1 to 6678)
  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: 
            "10.1261/rna.042838.113"; journalName: "RNA"; date: "2014-05"; 
            volume: "20"; issue: "5"; pages: "656-69"
COMMENT     Sequence Label: pET28b-sad1-H85A
FEATURES             Location/Qualifiers
     source          1..6678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             28..45
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             49..81
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1444..1461
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1514..1532)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1528..1575
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1612..2067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1699..1718)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1909..1930
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2159..2974)
                     /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(2159..2974)
                     /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     2882..2901
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2966..2985
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3096..3684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3585..3604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3838..3855
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3870..4009
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3935..3954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4095..4117
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4111..4302)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5111..6193)
                     /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     6155..6174
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6194..6271)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6477..6496
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6580..6599
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6580..6598
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6599..6623
                     /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             6638..6660
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat ggctagcatg
       61 actggtggac agcaaatggg tcgggatccc atggaggttg ataacaaaag gagacattca
      121 gaagatgagt taaaacagga ggcagtaaaa aagataaagt ctcaggaacc aaactatgcc
      181 tatcttgaga ctgtagtaag agaaaaacta gatttcgatt ctgagaaaat ttgctgcatt
      241 actctgtccc ctttgaatgt atattgttgc ctcgtgtgtg gacattatta tcaggggaga
      301 catgaaaaga gtcctgcttt catacattca attgatgaaa acgcgcacgt cttcttgaat
      361 ttgacaagtt tgaagtttta catgttgccg caaaatgtcc aaattttaca tgacggtgag
      421 gttcaattgt tgaacagcat aaaatttgca gcttatccga cttattgtcc aaaagatcta
      481 gaagattttc cgagacagtg ttttgatttg agcaatcgca catatctgaa tggatttata
      541 gggttcacca atgcggcaac ttatgactat gcgcatagtg tattgctttt gatatcgcac
      601 atggttccag tgcgggacca cttcctactg aatcattttg ataatcaagg ggaatttatc
      661 aaaaggttat ccatatgtgt aaagaaaatt tggtccccta aactattcaa acatcatctt
      721 tcagttgacg attttgtatc ctatttgaaa gttagagaag gtttaaattt aaatcctata
      781 gacccgcgtc tcttcctttt gtggctattc aataaaattt gctcgtcctc gaacgactta
      841 aagtcaattt taaaccattc ttgtaaaggc aaagtaaaga tagcaaaagt tgaaaataag
      901 cctgaggcca gtgagtctgt tacaggaaaa gtcattgtca aaccattttg ggttctgact
      961 ctcgatttgc cggagttttc accctttgaa gatggcaaca gcgttgatga tctcccacaa
     1021 ataaatatca ctaaattatt gactaaattt actaagtcaa gaagctcctc tactagcacc
     1081 gtttttgaac taacgcgatt accgcagttt ttaatatttc atttcaatag gtttgaccgt
     1141 aacagtgatc atcctgtcaa aaatagaaac caaacgttgg tggaattttc cagtgaactt
     1201 gaaattttgc acgtcaagta tcgcttaaaa gcaaatgtgg tccatgtggt cattaaacaa
     1261 ccatccacag atggaaacgc atttaatggt gatgaaaaaa gccattggat tactcagctg
     1321 tatgataaca aaagtgaaaa atggatagaa atagatggca taaatacgac agagagagaa
     1381 gccgagcttt tatttttgaa ggaaacgttt atacaagtat gggaaaagca agagtgactc
     1441 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     1501 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1561 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1621 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1681 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1741 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1801 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1861 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1921 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1981 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2041 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     2101 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     2161 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     2221 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     2281 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     2341 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     2401 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     2461 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     2521 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     2581 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     2641 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     2701 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     2761 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     2821 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     2881 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     2941 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     3001 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     3061 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3121 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3181 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3241 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3301 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3361 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3421 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3481 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3541 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3601 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3661 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3721 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3781 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3841 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3901 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3961 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     4021 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4081 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4141 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     4201 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4261 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4321 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4381 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4441 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4501 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4561 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4621 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4681 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4741 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4801 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4861 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     4921 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4981 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     5041 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     5101 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5161 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     5221 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     5281 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     5341 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     5401 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     5461 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     5521 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     5581 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     5641 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5701 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5761 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5821 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5881 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5941 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     6001 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     6061 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     6121 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     6181 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     6241 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     6301 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     6361 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     6421 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     6481 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     6541 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     6601 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
     6661 tataccatgg gcagcagc
//
