LOCUS       sequence-102305-        7209 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRS305 pPGK1-BirA::LEU2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7209)
  AUTHORS   Jan CH, Williams CC, Weissman JS
  TITLE     Principles of ER cotranslational translocation revealed by 
            proximity-specific ribosome profiling.
  JOURNAL   Science. 2014 Nov 7;346(6210):1257521. doi: 10.1126/science.1257521.
            Epub 2014 Nov 6.
  PUBMED    25378630
REFERENCE   2  (bases 1 to 7209)
  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: "Science."; 
            date: "2014-11-7"; volume: "346(6210):1257521. doi"; pages: " 
            10.1126/science.1257521. Epub 2014 Nov 6"
COMMENT     Sequence Label: pJW1234
FEATURES             Location/Qualifiers
     source          1..7209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(663..1757)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1770..2174)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2474..2929)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2611..2632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2823..2842
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3059..3081
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3073..3090
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3074..3090
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3097..3116
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3097..3115
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3549..4514
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3549..4511
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     terminator      4657..4904
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4673..4691)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(4949..4967)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4988..5004)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4988..5004)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5001..5023)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5012..5028
                     /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)."
     promoter        complement(join(5036..5042,5043..5060,5061..5066))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5219..5236)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5390..5978)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6149..6940,6941..7009))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6772..6791
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7010..7114)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7182..7200
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
      241 accattatgg gaaatggttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca
      301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat
      361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
      421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
      481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
      541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg
      601 tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct
      661 ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg
      721 ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct
      781 tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac
      841 aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat
      901 ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc
      961 aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg
     1021 ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca
     1081 gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc
     1141 acagtttttc tccataatct tgaagaggcc aaaacattag ctttatccaa ggaccaaata
     1201 ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact
     1261 tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc
     1321 ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca
     1381 aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt
     1441 aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca
     1501 ctaccggtac cccatttagg accacccaca gcacctaaca aaacggcatc aaccttcttg
     1561 gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca
     1621 attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga
     1681 accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc
     1741 ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata
     1801 tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat
     1861 tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat
     1921 ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct
     1981 ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca
     2041 aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat
     2101 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga
     2161 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg
     2221 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt
     2281 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg
     2341 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta
     2401 gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2461 ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
     2521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg
     2941 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     3001 cagctggcga aggggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     3061 cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa
     3121 ttggagctcc accgcggtgg cggccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3181 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3241 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3301 cgaaacgcgc gaggcagaga tcccgactct tttcttctaa ccaagggggt ggtttagttt
     3361 agtagaacct cgtgaaactt acatttacat atatataaac ttgcataaat tggtcaatgc
     3421 aagaaataca tatttggtct tttctaattc gtagtttttc aagttcttag atgctttctt
     3481 tttctctttt ttacagatca tcaaggaagt aattatctac tttttacaac aaatataaaa
     3541 caagatctat gaaggataac accgtgccac tgaaattgat tgccctgtta gcgaacggtg
     3601 aatttcactc tggcgagcag ttgggtgaaa cgctgggaat gagccgggcg gctattaata
     3661 aacacattca gacactgcgt gactggggcg ttgatgtctt taccgttccg ggtaaaggat
     3721 acagcctgcc tgagcctatc cagttactta atgctaaaca gatattgggt cagctggatg
     3781 gcggtagtgt agccgtgctg ccagtgattg actccacgaa tcagtacctt cttgatcgta
     3841 tcggagagct taaatcgggc gatgcttgca ttgcagaata ccagcaggct ggccgtggtc
     3901 gccggggtcg gaaatggttt tcgccttttg gcgcaaactt atatttgtcg atgttctggc
     3961 gtctggaaca aggcccggcg gcggcgattg gtttaagtct ggttatcggt atcgtgatgg
     4021 cggaagtatt acgcaagctg ggtgcagata aagttcgtgt taaatggcct aatgacctct
     4081 atctgcagga tcgcaagctg gcaggcattc tggtggagct gactggcaaa actggcgatg
     4141 cggcgcaaat agtcattgga gccgggatca acatggcaat gcgccgtgtt gaagagagtg
     4201 tcgttaatca ggggtggatc acgctgcagg aagcggggat caatctcgat cgtaatacgt
     4261 tggcggccat gctaatacgt gaattacgtg ctgcgttgga actcttcgaa caagaaggat
     4321 tggcacctta tctgtcgcgc tgggaaaagc tggataattt tattaatcgc ccagtgaaac
     4381 ttatcattgg tgataaagaa atatttggca tttcacgcgg aatagacaaa cagggggctt
     4441 tattacttga gcaggatgga ataataaaac cctggatggg cggtgaaata tccctgcgta
     4501 gtgcagaaaa ataatgacca tggcaatcac tagtcgaggg ggggcccggt accaagatgg
     4561 cctttggtgg gttgaagaag gaaaaagaca gaaacgactt aattacctac ttgaaaaaag
     4621 cctgtgagta aacaggcccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct
     4681 tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg
     4741 aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat
     4801 ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa
     4861 ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcaagctt gtcgacctcg
     4921 agggggggcc cggtaccagc ttttgttccc tttagtgagg gttaattccg agcttggcgt
     4981 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5041 taggagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagg taactcacat
     5101 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5161 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5221 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5281 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5341 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5401 tcggcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5461 caggactata aagataccag gcgttccccc ctggaagctc cctcgtgcgc tctcctgttc
     5521 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5581 ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5641 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5701 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5761 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5821 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5881 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5941 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6001 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6061 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6121 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6181 cagcgatctg tctatttcgt tcatccatag ttgcctgact gcccgtcgtg tagataacta
     6241 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6301 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6361 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6421 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6481 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6541 catgatcccc catgttgtga aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6601 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6661 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6721 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6781 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6841 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6901 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6961 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7021 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7081 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7141 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     7201 cctttcgtc
//
