LOCUS       sequence-178424-        7977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  yeast expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7977)
  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 7977)
  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: pRS313-Sad1-Wt
FEATURES             Location/Qualifiers
     source          1..7977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2273..2292)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2274..2292)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2299..2316)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2299..2315)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2308..2330)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2456..2911
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2543..2562)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2753..2774
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3171..3833)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(3834..4021)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(4168..4187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4287..4309
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4347..4365)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4413..4916
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4946..5050
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5051..5119,5120..5911)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5269..5288)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6082..6670
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6571..6590
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6824..6841
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6958..6979
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6994..6999,7000..7017,7018..7024)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7032..7048
                     /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     7037..7059
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7056..7072
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7056..7072
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7093..7111
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     7139..7155
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     7140..7156
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 atggaggttg ataacaaaag gagacattca gaagatgagt taaaacagga ggcagtaaaa
       61 aagataaagt ctcaggaacc aaactatgcc tatcttgaga ctgtagtaag agaaaaacta
      121 gatttcgatt ctgagaaaat ttgctgcatt actctgtccc ctttgaatgt atattgttgc
      181 ctcgtgtgtg gacattatta tcaggggaga catgaaaaga gtcctgcttt catacattca
      241 attgatgaaa accatcacgt cttcttgaat ttgacaagtt tgaagtttta catgttgccg
      301 caaaatgtcc aaattttaca tgacggtgag gttcaattgt tgaacagcat aaaatttgca
      361 gcttatccga cttattgtcc aaaagatcta gaagattttc cgagacagtg ttttgatttg
      421 agcaatcgca catatctgaa tggatttata gggttcacca atgcggcaac ttatgactat
      481 gcgcatagtg tattgctttt gatatcgcac atggttccag tgcgggacca cttcctactg
      541 aatcattttg ataatcaagg ggaatttatc aaaaggttat ccatatgtgt aaagaaaatt
      601 tggtccccta aactattcaa acatcatctt tcagttgacg attttgtatc ctatttgaaa
      661 gttagagaag gtttaaattt aaatcctata gacccgcgtc tcttcctttt gtggctattc
      721 aataaaattt gctcgtcctc gaacgactta aagtcaattt taaaccattc ttgtaaaggc
      781 aaagtaaaga tagcaaaagt tgaaaataag cctgaggcca gtgagtctgt tacaggaaaa
      841 gtcattgtca aaccattttg ggttctgact ctcgatttgc cggagttttc accctttgaa
      901 gatggcaaca gcgttgatga tctcccacaa ataaatatca ctaaattatt gactaaattt
      961 actaagtcaa gaagctcctc tactagcacc gtttttgaac taacgcgatt accgcagttt
     1021 ttaatatttc atttcaatag gtttgaccgt aacagtgatc atcctgtcaa aaatagaaac
     1081 caaacgttgg tggaattttc cagtgaactt gaaattttgc acgtcaagta tcgcttaaaa
     1141 gcaaatgtgg tccatgtggt cattaaacaa ccatccacag atggaaacgc atttaatggt
     1201 gatgaaaaaa gccattggat tactcagctg tatgataaca aaagtgaaaa atggatagaa
     1261 atagatggca taaatacgac agagagagaa gccgagcttt tatttttgaa ggaaacgttt
     1321 atacaagtat gggaaaagca agagtgaaag aaaagaaagt aaaaaaaaaa attggcactg
     1381 ggactaccag tagccatttc atttttactg ttatttaagg agccttttcc ctcctaacgc
     1441 gttttctctt tcccattcat tccattaact acttcattag aatttttaga aaaatacaat
     1501 ataatatata tgtagtgggc tttctagtta tttaatgcat aatgacttta taaaatttgt
     1561 ttatttaatt gccactgaat taacacccgc cgtcagcaca gaaacaatat tgttctctag
     1621 tgtctcatct gctgtttcgg aaaggtgctt ctttggatct tgcctaccaa cgtatcttgt
     1681 cttaagttct tcttcggtta attccttttc ctcttctatt ctcttagagt actgttcggc
     1741 tatcttaacc atactctttg tagcagccaa atttgattct tctttttctt cataatcgta
     1801 catcttaaga cctgattgcc actgttcttt atgtaagttc attaacatct tggtttcctt
     1861 cgcggtttta tgataatcaa tatttaaaga atagtaatgc ctattcagac cgtgaattaa
     1921 ggcttgaatg ttggccttgt tcaataagcc tgtgttggag gttgtttgtc taggctctaa
     1981 gttatttatc aatgcgcccg tgtcaatcaa tctaaaagca tcgatgacaa cttttccctt
     2041 aacggattga atagggtcaa caacgacagc aacagctctg ctgtttagtt gttcaaaaga
     2101 tttttgagta ttaacatcaa cagaagatag ccaacagcca aaccctggat gagagtggta
     2161 ccagccaacg accatttggt ctctgcccgt ttgttttaac atgtccatca tcttcgcttg
     2221 gaaaacatca tcgacagcct caacagaaac tccgcggtgg agctccaatt cgccctatag
     2281 tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     2341 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     2401 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg
     2461 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2521 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2581 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2641 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2701 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2761 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2821 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2881 aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
     2941 tgtgcggtat ttcacaccgc atagatccgt cgagttcaag agaaaaaaaa agaaaaagca
     3001 aaaagaaaaa aggaaagcgc gcctcgttca gaatgacacg tatagaatga tgcattacct
     3061 tgtcatcttc agtatcatac tgttcgtata catacttact gacattcata ggtatacata
     3121 tatacacatg tatatatatc gtatgctgca gctttaaata atcggtgtca ctacataaga
     3181 acacctttgg tggagggaac atcgttggta ccattgggcg aggtggcttc tcttatggca
     3241 accgcaagag ccttgaacgc actctcacta cggtgatgat cattcttgcc tcgcagacaa
     3301 tcaacgtgga gggtaattct gctagcctct gcaaagcttt caagaaaatg cgggatcatc
     3361 tcgcaagaga gatctcctac tttctccctt tgcaaaccaa gttcgacaac tgcgtacggc
     3421 ctgttcgaaa gatctaccac cgctctggaa agtgcctcat ccaaaggcgc aaatcctgat
     3481 ccaaaccttt ttactccacg cacggcccct agggcctctt taaaagcttg accgagagca
     3541 atcccgcagt cttcagtggt gtgatggtcg tctatgtgta agtcaccaat gcactcaacg
     3601 attagcgacc agccggaatg cttggccaga gcatgtatca tatggtccag aaaccctata
     3661 cctgtgtgga cgttaatcac ttgcgattgt gtggcctgtt ctgctactgc ttctgcctct
     3721 ttttctggga agatcgagtg ctctatcgct aggggaccac cctttaaaga gatcgcaatc
     3781 tgaatcttgg tttcatttgt aatacgcttt actagggctt tctgctctgt catctttgcc
     3841 ttcgtttatc ttgcctgctc attttttagt atattcttcg aagaaatcac attactttat
     3901 ataatgtata attcattatg tgataatgcc aatcgctaag aaaaaaaaag agtcatccgc
     3961 taggtggaaa aaaaaaaatg aaaatcatta ccgaggcata aaaaaatata gagtgtacta
     4021 gaggaggcca agagtaatag aaaaagaaaa ttgcgggaaa ggactgtgtt atgacttccc
     4081 tgactaatgc cgtgttcaaa cgatacctgg cagtgactcc tagcgctcac caagctctta
     4141 aaacgggaat ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4201 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4261 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4321 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4381 catgataata atggtttctt agggtccttt tcatcacgtg ctataaaaat aattataatt
     4441 taaatttttt aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa
     4501 aaaatagttt ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta
     4561 ccttttatct tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt
     4621 gtagaagacc acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt
     4681 atatctattt aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt
     4741 gaaatttttt aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt
     4801 tatttacttt ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc
     4861 caaattattc catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc
     4921 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4981 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5041 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5101 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5161 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5221 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5281 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5341 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5401 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5461 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5521 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5581 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5641 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5701 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5761 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5821 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5881 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5941 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6001 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6061 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6241 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6661 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6721 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     6781 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     6841 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     6901 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     6961 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     7021 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     7081 cgccaagctc gaaattaacc ctcactaaag ggaacaaaag ctggtaccgg gccccccctc
     7141 gaggtcgacg gtatcgataa gcttgatatc gaattcatca atgtcaaaca ctctcattgc
     7201 ctcatccaaa ctcaaaggca taccgctcca tatgacatct tcttcatcga tgtttggcaa
     7261 aaacaatgtc agcttatctg tgctacagtt aaacaatatc gccgaagcag gtatatcaac
     7321 acctgacaaa tgataaaaat atctattctg tctaaaatct ttgttggtgt cacaatactt
     7381 gtttccctct aattcttcac cagcaataaa aaatgctgtg gatatctttg atagcttcgg
     7441 attgcgattt aacaacaaat ctttaaccct caagttatgt tctttcgcag gatacttatg
     7501 atttcttatc ttgaccggaa tttcatttac gtttgctacc tcgcccattt cttccccttt
     7561 ttctttactg taataatact tatctttgta ctctggctgt aaaattcctc ttgttctttg
     7621 ccgttctcca cgtttgaaat acttcaccaa gccaaagaaa aacaccaggg atccgaaaac
     7681 aacaagagag attggttcaa ggcacatcac tccaacaaca gatcaggtat atacctatat
     7741 ttcccaggtt ttcctacagc tcgacctgga aaaaagatga cacagtttct gaagaatcgc
     7801 cgtagcggaa agtgtcaatt gaacctccct cgcgtatagt aagcaatgcc aaaaaaaaaa
     7861 aaaaactgga aaatcttgat aaaaaaatga cgaaatagga aaaaaaattg gcagcaatct
     7921 aaccatgctc ttcatggttc atcatttagt gggcgttaac ctaaagcgct gtttata
//
