LOCUS       sequence-182854-        6306 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 6306)
  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 6306)
  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-ZnF-UBPdel
FEATURES             Location/Qualifiers
     source          1..6306
                     /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             1171..1188
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1241..1259)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1255..1302
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1339..1794
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1426..1445)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1636..1657
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1886..2701)
                     /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(1886..2701)
                     /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     2609..2628
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2693..2712
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2823..3411
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3312..3331
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3565..3582
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3597..3736
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3662..3681)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3822..3844
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3838..4029)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4838..5920)
                     /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     5882..5901
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5921..5998)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6204..6223
                     /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 cgggatccct attgtccaaa agatctagaa gattttccga gacagtgttt tgatttgagc
      241 aatcgcacat atctgaatgg atttataggg ttcaccaatg cggcaactta tgactatgcg
      301 catagtgtat tgcttttgat atcgcacatg gttccagtgc gggaccactt cctactgaat
      361 cattttgata atcaagggga atttatcaaa aggttatcca tatgtgtaaa gaaaatttgg
      421 tcccctaaac tattcaaaca tcatctttca gttgacgatt ttgtatccta tttgaaagtt
      481 agagaaggtt taaatttaaa tcctatagac ccgcgtctct tccttttgtg gctattcaat
      541 aaaatttgct cgtcctcgaa cgacttaaag tcaattttaa accattcttg taaaggcaaa
      601 gtaaagatag caaaagttga aaataagcct gaggccagtg agtctgttac aggaaaagtc
      661 attgtcaaac cattttgggt tctgactctc gatttgccgg agttttcacc ctttgaagat
      721 ggcaacagcg ttgatgatct cccacaaata aatatcacta aattattgac taaatttact
      781 aagtcaagaa gctcctctac tagcaccgtt tttgaactaa cgcgattacc gcagttttta
      841 atatttcatt tcaataggtt tgaccgtaac agtgatcatc ctgtcaaaaa tagaaaccaa
      901 acgttggtgg aattttccag tgaacttgaa attttgcacg tcaagtatcg cttaaaagca
      961 aatgtggtcc atgtggtcat taaacaacca tccacagatg gaaacgcatt taatggtgat
     1021 gaaaaaagcc attggattac tcagctgtat gataacaaaa gtgaaaaatg gatagaaata
     1081 gatggcataa atacgacaga gagagaagcc gagcttttat ttttgaagga aacgtttata
     1141 caagtatggg aaaagcaaga gtgactcgag caccaccacc accaccactg agatccggct
     1201 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1261 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1321 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     1381 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     1441 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     1501 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     1561 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1621 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1681 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1741 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     1801 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1861 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     1921 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     1981 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     2041 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     2101 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     2161 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     2221 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     2281 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     2341 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     2401 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     2461 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     2521 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     2581 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     2641 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     2701 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     2761 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2821 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2881 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2941 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3001 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3061 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3121 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3181 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3241 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3301 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3361 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3421 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3481 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3541 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     3601 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     3661 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     3721 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3781 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3841 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     3901 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     3961 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4021 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4081 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4141 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4201 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4261 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4321 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     4381 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     4441 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     4501 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     4561 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     4621 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     4681 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     4741 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     4801 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4861 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4921 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     4981 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5041 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     5101 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     5161 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     5221 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     5281 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     5341 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     5401 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     5461 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     5521 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     5581 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     5641 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     5701 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     5761 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     5821 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     5881 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     5941 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     6001 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     6061 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     6121 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     6181 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     6241 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     6301 cgaaat
//
