LOCUS       sequence-335126-        6528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. cerevisiae (yeast) vector for constitutive expression of 
            3xFLAG-Nop4 delta Exon 8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6528)
  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 6528)
  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 dE8
FEATURES             Location/Qualifiers
     source          1..6528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     123..142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     376..393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    510..531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(546..551,552..569,570..576)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    584..600
                     /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     589..611
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     608..624
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     608..624
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     643..663
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        645..663
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        688..1331
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     1225..1248
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     1337..1353
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1337..1353
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1370..1435
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1436..1460
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      2263..2510
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2279..2297)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2528..2547)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2529..2547)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2557..2574)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2557..2573)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2566..2588)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2714..3169
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2801..2820)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3011..3032
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3272..3946)
                     /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(3947..4227)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4248..4267)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4367..4389
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4427..4445)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4486..4989
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5026..5130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5131..5199,5200..5991)
                     /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(5349..5368)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6162..6528,1..222)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
       61 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      361 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      421 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      481 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      541 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      601 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
      661 agggaacaaa agctggagct cagtttatca ttatcaatac tcgccatttc aaagaatacg
      721 taaataatta atagtagtga ttttcctaac tttatttagt caaaaaatta gccttttaat
      781 tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt acacagaata tataacatcg
      841 taggtgtctg ggtgaacagt ttattcctgg catccactaa atataatgga gcccgctttt
      901 taagctggca tccagaaaaa aaaagaatcc cagcaccaaa atattgtttt cttcaccaac
      961 catcagttca taggtccatt ctcttagcgc aactacagag aacaggggca caaacaggca
     1021 aaaaacgggc acaacctcaa tggagtgatg caacctgcct ggagtaaatg atgacacaag
     1081 gcaattgacc cacgcatgta tctatctcat tttcttacac cttctattac cttctgctct
     1141 ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag ttccctgaaa ttattcccct
     1201 acttgactaa taagtatata aagacggtag gtattgattg taattctgta aatctatttc
     1261 ttaaacttct taaattctac ttttatagtt agtctttttt ttagttttaa aacaccagaa
     1321 cttagtttcg acggattcta gaactagtgg atcccccggg ctgcagatgg actacaaaga
     1381 ccatgacggt gattataaag atcatgatat cgattacaag gatgacgatg acaagacaag
     1441 tttgtacaaa aaagcaggct acaaaatgga agaaacaata gaaaatgttg aagtgccttc
     1501 tagtaatgtc tcaaaacaaa atgacgacgg cctagatatg aagactttgt ttgtgaggtc
     1561 tataccccag gatgtcacag atgagcagct ggcagatttc ttttccaatt ttgctccgat
     1621 caagcatgcg gtagttgtta aggacacgaa taaaaggtcc aggggcttcg ggttcgttag
     1681 ttttgctgta gaggatgaca caaaagaagc cctagctaag gccagaaaga ccaaatttaa
     1741 cggacatatc ttgagggtag acattgctaa aagaagagat cgttcaaaga aaacatcaga
     1801 agtggttgaa aagagcacac ctgagtcatc tgagaaaata acgggccaga acaatgaaga
     1861 tgaggacgat gctgatggtg aagactccat gctaaaaggt aaaccgaaat taattatcag
     1921 aaatatgcca tggagttgtc gtgatccagt taaattgaaa aagatctttg gtagatatgg
     1981 taccgtcgtg gaggctacca ttccaaggaa gcgtgatggt aaattgtgtg gatttgcgtt
     2041 tgttactatg aagaaaatca gtaattgtag aattgctttg gaaaacacca aagacttgaa
     2101 aattgatggt agaaaggtgg ctgttgattt tgctgttcaa aaaaacagat gggaagatta
     2161 caaaaaagcc cagccagaaa tgaatgacaa agatgataac gagtcaggaa atgaagatgc
     2221 tgaagaaaac catgacgacg aagaagatga aaatgaatag agtcatgtaa ttagttatgt
     2281 cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac
     2341 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
     2401 ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac
     2461 tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc ggccggtacc
     2521 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     2581 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     2641 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     2701 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2761 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2821 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2881 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2941 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3001 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3061 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3121 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt
     3181 attttctcct tacgcatctg tgcggtattt cacaccgcat agatcggcaa gtgcacaaac
     3241 aatacttaaa taaatactac tcagtaataa cctatttctt agcatttttg acgaaatttg
     3301 ctattttgtt agagtctttt acaccatttg tctccacacc tccgcttaca tcaacaccaa
     3361 taacgccatt taatctaagc gcatcaccaa cattttctgg cgtcagtcca ccagctaaca
     3421 taaaatgtaa gctttcgggg ctctcttgcc ttccaaccca gtcagaaatc gagttccaat
     3481 ccaaaagttc acctgtccca cctgcttctg aatcaaacaa gggaataaac gaatgaggtt
     3541 tctgtgaagc tgcactgagt agtatgttgc agtcttttgg aaatacgagt cttttaataa
     3601 ctggcaaacc gaggaactct tggtattctt gccacgactc atctccatgc agttggacga
     3661 tatcaatgcc gtaatcattg accagagcca aaacatcctc cttaggttga ttacgaaaca
     3721 cgccaaccaa gtatttcgga gtgcctgaac tatttttata tgcttttaca agacttgaaa
     3781 ttttccttgc aataaccggg tcaattgttc tctttctatt gggcacacat ataataccca
     3841 gcaagtcagc atcggaatct agagcacatt ctgcggcctc tgtgctctgc aagccgcaaa
     3901 ctttcaccaa tggaccagaa ctacctgtga aattaataac agacatactc caagctgcct
     3961 ttgtgtgctt aatcacgtat actcacgtgc tcaatagtca ccaatgccct ccctcttggc
     4021 cctctccttt tcttttttcg accgaattaa ttcttaatcg gcaaaaaaag aaaagctccg
     4081 gatcaagatt gtacgtaagg tgacaagcta tttttcaata aagaatatct tccactactg
     4141 ccatctggcg tcataactgc aaagtacaca tatattacga tgctgttcta ttaaatgctt
     4201 cctatattat atatatagta atgtcgtgat ctatggtgca ctctcagtac aatctgctct
     4261 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4321 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4381 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     4441 ctatttttat aggttaatgt catgataata atggtttctt agacggatcg cttgcctgta
     4501 acttacacgc gcctcgtatc ttttaatgat ggaataattt gggaatttac tctgtgttta
     4561 tttattttta tgttttgtat ttggatttta gaaagtaaat aaagaaggta gaagagttac
     4621 ggaatgaaga aaaaaaaata aacaaaggtt taaaaaattt caacaaaaag cgtactttac
     4681 atatatattt attagacaag aaaagcagat taaatagata tacattcgat taacgataag
     4741 taaaatgtaa aatcacagga ttttcgtgtg tggtcttcta cacagacaag atgaaacaat
     4801 tcggcattaa tacctgagag caggaagagc aagataaaag gtagtatttg ttggcgatcc
     4861 ccctagagtc ttttacatct tcggaaaaca aaaactattt tttctttaat ttcttttttt
     4921 actttctatt tttaatttat atatttatat taaaaaattt aaattataat tatttttata
     4981 gcacgtgatg aaaaggaccc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5041 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5101 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5161 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5221 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5281 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5341 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5401 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5461 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5521 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5581 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5641 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5701 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5761 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5821 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5881 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5941 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6001 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6061 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6121 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6181 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6241 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6301 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6361 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6421 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6481 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacacc
//
