LOCUS       sequence-182853-        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-D422A
FEATURES             Location/Qualifiers
     source          1..6678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             148..180
                     /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             1543..1560
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1613..1631)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1627..1674
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1711..2166
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1798..1817)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2008..2029
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2258..3073)
                     /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(2258..3073)
                     /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     2981..3000
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3065..3084
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3195..3783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3684..3703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3937..3954
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3969..4108
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4034..4053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4194..4216
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4210..4401)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5210..6292)
                     /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     6254..6273
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6293..6370)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6576..6595
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcatga ctggtggaca gcaaatgggt
      181 cgggatccca tggaggttga taacaaaagg agacattcag aagatgagtt aaaacaggag
      241 gcagtaaaaa agataaagtc tcaggaacca aactatgcct atcttgagac tgtagtaaga
      301 gaaaaactag atttcgattc tgagaaaatt tgctgcatta ctctgtcccc tttgaatgta
      361 tattgttgcc tcgtgtgtgg acattattat caggggagac atgaaaagag tcctgctttc
      421 atacattcaa ttgatgaaaa ccatcacgtc ttcttgaatt tgacaagttt gaagttttac
      481 atgttgccgc aaaatgtcca aattttacat gacggtgagg ttcaattgtt gaacagcata
      541 aaatttgcag cttatccgac ttattgtcca aaagatctag aagattttcc gagacagtgt
      601 tttgatttga gcaatcgcac atatctgaat ggatttatag ggttcaccaa tgcggcaact
      661 tatgactatg cgcatagtgt attgcttttg atatcgcaca tggttccagt gcgggaccac
      721 ttcctactga atcattttga taatcaaggg gaatttatca aaaggttatc catatgtgta
      781 aagaaaattt ggtcccctaa actattcaaa catcatcttt cagttgacga ttttgtatcc
      841 tatttgaaag ttagagaagg tttaaattta aatcctatag acccgcgtct cttccttttg
      901 tggctattca ataaaatttg ctcgtcctcg aacgacttaa agtcaatttt aaaccattct
      961 tgtaaaggca aagtaaagat agcaaaagtt gaaaataagc ctgaggccag tgagtctgtt
     1021 acaggaaaag tcattgtcaa accattttgg gttctgactc tcgatttgcc ggagttttca
     1081 ccctttgaag atggcaacag cgttgatgat ctcccacaaa taaatatcac taaattattg
     1141 actaaattta ctaagtcaag aagctcctct actagcaccg tttttgaact aacgcgatta
     1201 ccgcagtttt taatatttca tttcaatagg tttgaccgta acagtgatca tcctgtcaaa
     1261 aatagaaacc aaacgttggt ggaattttcc agtgaacttg aaattttgca cgtcaagtat
     1321 cgcttaaaag caaatgtggt ccatgtggtc attaaacaac catccacaga tggaaacgca
     1381 tttaatggtg atgaaaaaag ccattggatt actcagctgt atgataacaa aagtgaaaaa
     1441 tggatagaaa tagctggcat aaatacgaca gagagagaag ccgagctttt atttttgaag
     1501 gaaacgttta tacaagtatg ggaaaagcaa gagtgactcg agcaccacca ccaccaccac
     1561 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1621 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1681 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     1741 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1801 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1861 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1921 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1981 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2041 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2101 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     2161 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2221 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     2281 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     2341 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     2401 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     2461 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     2521 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     2581 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     2641 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     2701 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     2761 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2821 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2881 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2941 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     3001 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     3061 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     3121 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3181 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3241 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3301 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3361 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3421 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3481 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3541 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3601 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3661 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3721 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3781 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3841 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3901 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3961 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     4021 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4081 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4141 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4201 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4261 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4321 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4381 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4441 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     4501 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4561 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4621 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4681 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     4741 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     4801 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     4861 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     4921 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     4981 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     5041 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     5101 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     5161 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     5221 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5281 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     5341 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5401 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5461 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     5521 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5581 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     5641 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     5701 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     5761 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     5821 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     5881 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5941 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     6001 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     6061 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     6121 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     6181 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     6241 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     6301 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     6361 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     6421 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     6481 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     6541 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6601 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     6661 atctcgatcc cgcgaaat
//
