LOCUS       sequence-225411-        6216 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses K55M RPL28 from HIS3 yeast plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6216)
  AUTHORS   McGeachy AM, Meacham ZA, Ingolia NT
  TITLE     An Accessible Continuous-Culture Turbidostat for Pooled Analysis of 
            Complex Libraries.
  JOURNAL   ACS Synth Biol. 2019 Apr 19;8(4):844-856. doi: 
            10.1021/acssynbio.8b00529. Epub 2019 Apr 2.
  PUBMED    30908907
REFERENCE   2  (bases 1 to 6216)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.8b00529"; journalName: "ACS Synth Biol"; date: 
            "2019-04-19- 19"; volume: "8"; issue: "4"; pages: "844-856"
COMMENT     Sequence Label: pRPL28(K55M) SpHIS5 (pNTI666)
FEATURES             Location/Qualifiers
     source          1..6216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..61)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(215..803)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(295..314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(974..1765,1766..1834))
                     /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     1597..1616
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1835..1939)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2007..2025
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2063..2085)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2185..2204
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2285..2303
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2285..2302
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    2347..2850
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     terminator      4588..4775
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4643..4667)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            4850..6050
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        4850..5193
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             5194..5847
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S. 
                     cerevisiae HIS3"
                     /translation="MGRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHTNQ
                     KGEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQ
                     AMSNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLY
                     SFSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      5853..6050
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(6154..6173)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6155..6173)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
       61 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
      121 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
      181 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
      241 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
      301 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
      361 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
      421 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
      481 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
      541 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
      601 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
      661 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
      721 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
      781 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
      841 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
      901 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
      961 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     1021 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     1081 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     1141 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     1201 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     1261 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     1321 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     1381 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     1441 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     1501 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     1561 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     1621 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     1681 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     1741 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     1801 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     1861 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     1921 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     1981 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     2041 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     2101 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     2161 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     2221 tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca
     2281 tacgatttag gtgacactat agaacgcggc cgccagctga agcttcgtac gctgcaggtc
     2341 cttttcatca cgtgctataa aaataattat aatttaaatt ttttaatata aatatataaa
     2401 ttaaaaatag aaagtaaaaa aagaaattaa agaaaaaata gtttttgttt tccgaagatg
     2461 taaaagactc tagggggatc gccaacaaat actacctttt atcttgctct tcctgctctc
     2521 aggtattaat gccgaattgt ttcatcttgt ctgtgtagaa gaccacacac gaaaatcctg
     2581 tgattttaca ttttacttat cgttaatcga atgtatatct atttaatctg cttttcttgt
     2641 ctaataaata tatatgtaaa gtacgctttt tgttgaaatt ttttaaacct ttgtttattt
     2701 ttttttcttc attccgtaac tcttctacct tctttattta ctttctaaaa tccaaataca
     2761 aaacataaaa ataaataaac acagagtaaa ttcccaaatt attccatcat taaaagatac
     2821 gaggcgcgtg taagttacag gcaagcgatc cgtccggatc caaattatgc caccgttacc
     2881 ttacgtttca tggttaatca tcgtttactg ccgcctatga gcgtaagcta atgttataaa
     2941 gaaacaagct ataatattgt taaatatagt tgatcaacag cattgtaatg attacaagag
     3001 acgaggtgga atgaacctta tgaaatgcgt attatatata aactgtaata agagctaagt
     3061 tgaattgaaa tctacgatac ttgatgttga cattatagca ctagttccca ggaaaccctt
     3121 tcgaaaaaca cagcaaaaac aagagtactg taaccaatgt aacatctgta caccagggac
     3181 ccacacatta ccaaaatcaa aattattttt ctaatgcctg ttatttttcc tatttttcct
     3241 ctggcgcgtg aatagcccgc agagacgcaa acaattttcc tcgcagtttt tcgcttgttt
     3301 aatgcgtatt ttcccagata ggttcaaacc tttcatctgt atcccgtata tttaagatgg
     3361 cgtttgcttt ctccgttgat ttttttcctt cttagtgatt tttttgcatt aaatcccaga
     3421 acaatcatcc aactaatcaa gaatgccttc cagattcact aagactagaa agcacagagg
     3481 tcacgtctca ggtatgtagt tccatttgga agagggaatg aaagaaccaa gacggtgact
     3541 ttttttttag tgttgtgcaa ccaatatgtc gtgtgtatat catggtacag gagaatgtca
     3601 atcagctaag tgtactcaac atatttcttt gtgttttgat tgcgaacttt gtattaccat
     3661 ctcactgttg agacggctta tttgaggtaa tagctcgagt aaatgtactc ttccatcgca
     3721 aactgagcaa aaagaaagtg tgcatagcct ttgtcatact tctcctttat tataccatga
     3781 tattcagaac agtcatactg tctactcatt ttacggctat aaaaggtaac tttcatttag
     3841 attatggaaa gcactaatta tcgctgtatc aattgaaatg gttgtagaga gcgcaattat
     3901 gaaaaagagt taccacgttt cttttgtttc gataaaatgt ccagttgaaa acctgtttta
     3961 ctaacgattt aaaaattgta tttcattaca atattttttt tgtacagccg gtaaaggtcg
     4021 tatcggtaag cacagaaagc accccggtgg tagaggtatg gccggtggtc aacatcacca
     4081 cagaattaac atggataaat accatccagg ttatttcggt atggttggta tgagatactt
     4141 ccacaagcaa caagctcatt tctggaagcc agtcttgaac ttggacaaat tgtggacatt
     4201 gatcccagaa gacaagagag accaatactt gaaatctgct tctaaggaaa ctgctccagt
     4261 tattgacact ttggcagccg gttacggtaa gatcttgggt aagggtagaa ttccaaatgt
     4321 tccagttatc gtcaaagcta gattcgtctc caagttggct gaagaaaaaa tcagagctgc
     4381 tggtggtgtt gttgaattga tcgcttaagc gcatcaacaa aaactctatg tattttccaa
     4441 taaattatat atcttcagtt taatctaatt caacatctac ttctgtatta tttctatcac
     4501 ccattttcac cgttttttcg tcttgtctat tttgaagtta ccagtggcag atcccaacgc
     4561 ttttatcgcg ctagcacttc taaataagcg aatttcttat gatttatgat ttttattatt
     4621 aaataagtta taaaaaaaat aagtgtatac aaattttaaa gtgactctta ggttttaaaa
     4681 cgaaaattct tattcttgag taactctttc ctgtaggtca ggttgctttc tcaggtatag
     4741 tatgaggtcg ctcttattga ccacacctct accggcagat ccgctaggga taacagggta
     4801 atatagatct gtttagcttg cctcgtcccc gccgggtcac ccggccagcg acatggaggc
     4861 ccagaatacc ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg
     4921 cccgtacatt tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg
     4981 ccgcacggcg cgaagcaaaa attacggctc ctcgctgcag acctgcgagc agggaaacgc
     5041 tcccctcaca gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg
     5101 ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata
     5161 cagttctcac atcacatccg aacataaaca accatgggta ggagggcttt tgtagaaaga
     5221 aatacgaacg aaacgaaaat cagcgttgcc atcgctttgg acaaagctcc cttacctgaa
     5281 gagtcgaatt ttattgatga acttataact tccaagcata caaaccaaaa gggagaacaa
     5341 gtaatccaag tagacacggg aattggattc ttggatcaca tgtatcatgc actggctaaa
     5401 catgcaggct ggagcttacg actttactca agaggtgatt taatcatcga tgatcatcac
     5461 actgcagaag atactgctat tgcacttggt attgcattca agcaggctat gagtaacttt
     5521 gccggcgtta aaagatttgg acatgcttat tgtccacttg acgaagctct ttctagaagc
     5581 gtagttgact tgtcgggacg gccctatgct gttatcgatt tgggattaaa gcgtgaaaag
     5641 gttggggaat tgtcctgtga aatgatccct cacttactat attccttttc ggtagcagct
     5701 ggaattactt tgcatgttac ctgcttatat ggtagtaatg accatcatcg tgctgaaagc
     5761 gcttttaaat ctctggctgt tgccatgcgc gcggctacta gtcttactgg aagttctgaa
     5821 gtcccaagca cgaagggagt gttgtaaaga gtactgacaa taaaaagatt cttgttttca
     5881 agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta
     5941 gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc
     6001 agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc
     6061 gatactaacg ccgccatcca gtttaaacga gctcgaattc atcgatgata tcagatccac
     6121 tagtggccta tgcggccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga
     6181 taagccaggt taacctgcat taatgaatcg gccaac
//
