LOCUS       sequence-335122-        7814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. cerevisiae (yeast) vector for constitutive expression of 
            3xFLAG-Nop4 L306P.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7814)
  AUTHORS   Bryant CJ, Lorea CF, de Almeida HL Jr, Weinert L, Vedolin L, Pinto E
            Vairo F, Baserga SJ
  TITLE     Biallelic splicing variants in the nucleolar 60S assembly factor 
            RBM28 cause the ribosomopathy ANE syndrome.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 May 11;118(19). pii: 2017777118. doi:
            10.1073/pnas.2017777118.
  PUBMED    33941690
REFERENCE   2  (bases 1 to 7814)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2021-05-11"; volume: "118(19). pii: 
            2017777118. doi"; pages: " 10.1073/pnas.2017777118"
COMMENT     Sequence Label: p414GPD-3xFLAG-Nop4 L306P
FEATURES             Location/Qualifiers
     source          1..7814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     120..139
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     373..390
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    507..528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(543..548,549..566,567..573)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    581..597
                     /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     586..608
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     605..621
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     605..621
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     640..660
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        642..660
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        685..1328
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     1222..1245
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     1334..1350
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1334..1350
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1367..1432
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1433..1457
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3519..3543)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      3546..3793
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3562..3580)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3811..3830)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3812..3830)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3840..3857)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3840..3856)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3849..3871)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3997..4452
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4084..4103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4294..4315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4555..5229)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(5230..5510)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(5531..5550)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5650..5672
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5710..5728)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5769..6272
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6309..6413
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6414..6482,6483..7274)
                     /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(6632..6651)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7445..7814,1..219)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
       61 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
      121 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
      181 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
      241 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
      301 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
      361 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
      421 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
      481 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      541 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      601 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
      661 gaacaaaagc tggagctcag tttatcatta tcaatactcg ccatttcaaa gaatacgtaa
      721 ataattaata gtagtgattt tcctaacttt atttagtcaa aaaattagcc ttttaattct
      781 gctgtaaccc gtacatgccc aaaatagggg gcgggttaca cagaatatat aacatcgtag
      841 gtgtctgggt gaacagttta ttcctggcat ccactaaata taatggagcc cgctttttaa
      901 gctggcatcc agaaaaaaaa agaatcccag caccaaaata ttgttttctt caccaaccat
      961 cagttcatag gtccattctc ttagcgcaac tacagagaac aggggcacaa acaggcaaaa
     1021 aacgggcaca acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg acacaaggca
     1081 attgacccac gcatgtatct atctcatttt cttacacctt ctattacctt ctgctctctc
     1141 tgatttggaa aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta ttcccctact
     1201 tgactaataa gtatataaag acggtaggta ttgattgtaa ttctgtaaat ctatttctta
     1261 aacttcttaa attctacttt tatagttagt ctttttttta gttttaaaac accagaactt
     1321 agtttcgacg gattctagaa ctagtggatc ccccgggctg cagatggact acaaagacca
     1381 tgacggtgat tataaagatc atgatatcga ttacaaggat gacgatgaca agacaagttt
     1441 gtacaaaaaa gcaggctaca aaatggaaga aacaatagaa aatgttgaag tgccttctag
     1501 taatgtctca aaacaaaatg acgacggcct agatatgaag actttgtttg tgaggtctat
     1561 accccaggat gtcacagatg agcagctggc agatttcttt tccaattttg ctccgatcaa
     1621 gcatgcggta gttgttaagg acacgaataa aaggtccagg ggcttcgggt tcgttagttt
     1681 tgctgtagag gatgacacaa aagaagccct agctaaggcc agaaagacca aatttaacgg
     1741 acatatcttg agggtagaca ttgctaaaag aagagatcgt tcaaagaaaa catcagaagt
     1801 ggttgaaaag agcacacctg agtcatctga gaaaataacg ggccagaaca atgaagatga
     1861 ggacgatgct gatggtgaag actccatgct aaaaggtaaa ccgaaattaa ttatcagaaa
     1921 tatgccatgg agttgtcgtg atccagttaa attgaaaaag atctttggta gatatggtac
     1981 cgtcgtggag gctaccattc caaggaagcg tgatggtaaa ttgtgtggat ttgcgtttgt
     2041 tactatgaag aaaatcagta attgtagaat tgctttggaa aacaccaaag acttgaaaat
     2101 tgatggtaga aaggtggctg ttgattttgc tgttcaaaaa aacagatggg aagattacaa
     2161 aaaagcccag ccagaaatga atgacaaaga tgataacgag tcaggaaatg aagatgctga
     2221 agaaaaccat gacgacgaag aagatgaaaa tgaagaggaa gatagacaag tagatcaagc
     2281 ttcaaaaaac aaagaatcga aacgtaaagc acaaaacaaa agggaagatt tttctgtttt
     2341 tgttcgtaac gtcccttatg atgccactga agaatctccg gctccgcact tcagtaaatt
     2401 tggcagcgtt aaatacgcat tacctgttat cgataagtct acggggttgg ctaaaggtac
     2461 ggcctttgta gccttcaaag accaatatac atataacgag tgtatcaaaa atgcgcctgc
     2521 ggctggctct acctctttat tgataggtga tgatgtcatg cctgagtatg tgtatgaagg
     2581 tcgtgttctg tccattactc caacgttagt aagagaggat gctggaagaa tggcagaaaa
     2641 gaacgccgcc aaaagaaagg aggccctagg taaagcgcca ggcgaaaaag atagacgtaa
     2701 cttatatttg ctgaatgaag gtagagttgt tgaaggctca aaaatggctg atttgttaac
     2761 caacactgac atggagatca gagaaaaatc ttacaaatta agagtggagc aactgaaaaa
     2821 gaacccatct ttgcatttgt cgatgactag gttggctatc agaaatcttc ctagagcaat
     2881 gaatgataaa gcattaaagg cgttagctcg taaagctgtc gtggaatttg ctacagaagt
     2941 taaaaataag gaaagacatc cattgagtaa agaagagatt atcagatcta caaaggaaaa
     3001 atacaagttc atgggtccag atgagattga agctcaaaaa aagaaagaca aaaaaagtgg
     3061 tgttgtcaag caagcaaaag ttataatgga agttaaagga tcaactgcgg gaagaagtag
     3121 aggttacggt tttgtagaat tcagagatca taaaaacgct ttgatgggct taaggtggtt
     3181 gaattgtcat gccgttacct ccgatgaaat tttagagggt ttgaatgatg atgaaaagaa
     3241 acaagtagac aatgacttag gcaaaggaag acgtctttgt gttgaatttg cgattgagaa
     3301 ctctaacgtc gtaaagagaa gaagggaaca attgaagcaa gcaagaacca agagaacaag
     3361 accagataat gaagatactg gagatgtcgg agaatccgag aataagaagc caaaaaaaga
     3421 agaagcaacg acgcccacca atcccgatga caaaaagatg ggcgatgata taaagagaat
     3481 aatcgggttc aagcgtaaga gaaagcacgc aaaaaaaaac ccagctttct tgtacaaagt
     3541 ggtagtcatg taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct
     3601 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt
     3661 tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg
     3721 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa
     3781 ggctttaatt tgcggccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     3841 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     3901 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     3961 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
     4021 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4081 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4141 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4201 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4261 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4321 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     4381 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4441 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4501 catagatcgg caagtgcaca aacaatactt aaataaatac tactcagtaa taacctattt
     4561 cttagcattt ttgacgaaat ttgctatttt gttagagtct tttacaccat ttgtctccac
     4621 acctccgctt acatcaacac caataacgcc atttaatcta agcgcatcac caacattttc
     4681 tggcgtcagt ccaccagcta acataaaatg taagctttcg gggctctctt gccttccaac
     4741 ccagtcagaa atcgagttcc aatccaaaag ttcacctgtc ccacctgctt ctgaatcaaa
     4801 caagggaata aacgaatgag gtttctgtga agctgcactg agtagtatgt tgcagtcttt
     4861 tggaaatacg agtcttttaa taactggcaa accgaggaac tcttggtatt cttgccacga
     4921 ctcatctcca tgcagttgga cgatatcaat gccgtaatca ttgaccagag ccaaaacatc
     4981 ctccttaggt tgattacgaa acacgccaac caagtatttc ggagtgcctg aactattttt
     5041 atatgctttt acaagacttg aaattttcct tgcaataacc gggtcaattg ttctctttct
     5101 attgggcaca catataatac ccagcaagtc agcatcggaa tctagagcac attctgcggc
     5161 ctctgtgctc tgcaagccgc aaactttcac caatggacca gaactacctg tgaaattaat
     5221 aacagacata ctccaagctg cctttgtgtg cttaatcacg tatactcacg tgctcaatag
     5281 tcaccaatgc cctccctctt ggccctctcc ttttcttttt tcgaccgaat taattcttaa
     5341 tcggcaaaaa aagaaaagct ccggatcaag attgtacgta aggtgacaag ctatttttca
     5401 ataaagaata tcttccacta ctgccatctg gcgtcataac tgcaaagtac acatatatta
     5461 cgatgctgtt ctattaaatg cttcctatat tatatatata gtaatgtcgt gatctatggt
     5521 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5581 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5641 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5701 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5761 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
     5821 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
     5881 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
     5941 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
     6001 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
     6061 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
     6121 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
     6181 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
     6241 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
     6301 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6361 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6421 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6481 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6541 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6601 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6661 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6721 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6781 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6841 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6901 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6961 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     7021 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     7081 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     7141 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     7201 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7261 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7321 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7381 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7441 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7501 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7561 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     7621 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7681 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7741 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7801 cgacctacac cgaa
//
