LOCUS       sequence-482088-        6820 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Over-express Sc FLAG-POL4 in yeast (integrated).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6820)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 6820)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Sc FLAG-POL4-pRS404/GAL-L
FEATURES             Location/Qualifiers
     source          1..6820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(60..341)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(362..381)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     481..503
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(541..559)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        627..731
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(732..800,801..1592)
                     /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(950..969)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1763..2351
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2252..2271
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2505..2522
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2639..2660
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2675..2680,2681..2698,2699..2705)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2713..2729
                     /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     2718..2740
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2737..2753
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2737..2753
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2772..2792
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2774..2792
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(4650..4715)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        4728..5392
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     primer_bind     complement(4741..4764)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    5059..5176
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     complement(5420..5436)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5421..5437)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(5461..5480)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5462..5480)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5490..5507)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5490..5506)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5499..5521)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5647..6102
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5734..5753)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5944..5965
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(6205..6820,1..59))
                     /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"
ORIGIN
        1 tgcaagccgc aaactttcac caatggacca gaactacctg tgaaattaat aacagacata
       61 ctccaagctg cctttgtgtg cttaatcacg tatactcacg tgctcaatag tcaccaatgc
      121 cctccctctt ggccctctcc ttttcttttt tcgaccgaat taattcttaa tcggcaaaaa
      181 aagaaaagct ccggatcaag attgtacgta aggtgacaag ctatttttca ataaagaata
      241 tcttccacta ctgccatctg gcgtcataac tgcaaagtac acatatatta cgatgctgtt
      301 ctattaaatg cttcctatat tatatatata gtaatgtcgt tgatctatgg tgcactctca
      361 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
      421 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
      481 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
      541 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
      601 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
      661 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      721 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
      781 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
      841 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
      901 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
      961 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1021 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1081 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1141 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1201 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1261 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1321 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1381 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     1441 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     1501 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     1561 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1621 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     1681 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1741 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1801 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1861 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     1921 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     1981 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2041 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2101 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2161 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2221 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2281 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2341 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2401 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2461 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     2521 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     2581 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     2641 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     2701 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     2761 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggtg
     2821 gcggccgctc agcggtggca gcagccaact cagcttcctt tcgggctttg ttagcagccg
     2881 gatccttatg cagttttttt ttcccattcg atatttctat gttcgggttc agcgtatttt
     2941 aagtttaata actcgaaaat tctgcgttcg ttaaagcttt cgagaaggat attatttcga
     3001 aataaaccgt gttgtgtaag cttgaagcct ttttgcgctg ccaatattct tatccatcta
     3061 ttgtactctt tagatccagt atagtgtatt cttcctgctc caagctcatc ccacttgcaa
     3121 caaaaaaagt ctaatcttct gcaataattt ccatccttgg cattcagaga catatattgg
     3181 tcaatcggtt ttaattttgt ttcttcttct tcttcttctt caattaacaa tgtgccttca
     3241 tcatttttca tttctttcag ttcttctaat tctcttggag acaatttgac tcccatgaaa
     3301 aatttcttga ttatctcaga agaataccac ctctttcttt ctccataccc aacaatcttc
     3361 gctgtacgaa atctctccac tattcttttt aagaatagct tttccaagtt tggcgttagc
     3421 tgtaaaaaac aatggatata gccatccttg tacaacttaa tacaaagcgt ttccatgatt
     3481 tttgccaact cggtcgtgtc attacaaaac ggcttgaaaa ataaaagatc aatgtcacca
     3541 cacttggaat agcccctatt ataacttccc tgtaattcga cttggcattc aggatcaatg
     3601 ccacgcagcg ctttttgaac cttctttaaa tgtgtgaaac attcattccg agacatcttg
     3661 cataaccaat cgtcgtaata tgaccaacca aataaaattg tccaatctga tacaaactcc
     3721 tctgggtcct tcttagctgc aacacaaaag ctttcaaaat ttagaagatt ccagcgttta
     3781 gcaatttccg acccaatgcc gtaacaattt ttgaagtatt tcaacttgtc ttctaatccc
     3841 actgaatcat ttaaacctgg taaaactccc gtatctaata taacttgtat ttttttggca
     3901 atactaggcc cgatattcct taatttagta tgtgcttctt caccggaacg aacattgaaa
     3961 tcgcaatttt ccatcgactg tttagccagt ctataacttc ttgcacgaaa tttctcacct
     4021 tcgatctcat atttttttgt taaccttttc aacgctccga taatcaattc attgtttttg
     4081 tacttcgttt tgtcagtaac aagggaagct cttcctttat ccccctgtaa gagtcgctta
     4141 agaggttttg aagcttgtat catatcttta ttgttgctgt catcattcct ttcactttct
     4201 acgtcagttg aaatctcact ctctttatca gtagaacttt gtccgtcatc agcgttatct
     4261 gatataacga taatggatgg ttgaaattta atcaaatcat ctttttgaaa cgcaaattta
     4321 tcattctgga cccactttgt gatgcaactg gttttcacac attgaatgcc tgactgttca
     4381 atcttaccaa gaaattcatc gacatcattt aagcctgctt ccctttggaa aatatctttc
     4441 tgagtcaaat gcatatcaga atcaacaaag gagtcctcaa tcaaaatgac cacttcctta
     4501 cggctagatt gaagacaatt ttgaatactt gacacaattg tggcgccctt tttctccaat
     4561 atactcttaa agaacttatt ggaagatgtg ttaggattag gtaaaaaggc gaaaaattta
     4621 ccctttagag acatatgact agtggatcgc ttgtcatcgt catccttgta gtcgatgtca
     4681 tgatctttat aatcaccgtc atggtctttg tagtccatgg atccggggtt ttttctcctt
     4741 gacgttaaag tatagaggta tattaacaat tttttgttga tacttttatt acatttgaat
     4801 aagaagtaat acaaaccgaa aatgttgaaa gtattagtta aagtggttat gcagtttttg
     4861 catttatata tctgttaata gatcaaaaat catcgcttcg ctgattaatt accccagaaa
     4921 taaggctaaa aaactaatcg cattatcatc ctatggttgt taatttgatt cgttcatttg
     4981 aaggtttgtg gggccaggtt actgccaatt tttcctcttc ataaccataa aagctagtat
     5041 tgtagaatct ttattgttcg gagcagtgcg gcgcgaggca catctgcgtt tcaggaacgc
     5101 gaccggtgaa gacgaggacg cacggaggag agtcttcctt cggagggctg tcacccgctc
     5161 ggcggcttct aatccgtact tcaatatagc aatgagcagt taagcgtatt actgaaagtt
     5221 ccaaagagaa ggttttttta ggctaagata atggggctct ttacatttcc acaacatata
     5281 agtaagatta gatatggata tgtatatgga tatgtatatg gtggtaatgc catgtaatat
     5341 gattattaaa cttctttgcg tccatccaaa aaaaaagtaa gaatttttga aaattcaagg
     5401 aattcgatat caagcttatc gataccgtcg acctcgaggg ggggcccggt acccaattcg
     5461 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     5521 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5581 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     5641 gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5701 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5761 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5821 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5881 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5941 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     6001 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     6061 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct
     6121 ccttacgcat ctgtgcggta tttcacaccg catagatcgg caagtgcaca aacaatactt
     6181 aaataaatac tactcagtaa taacctattt cttagcattt ttgacgaaat ttgctatttt
     6241 gttagagtct tttacaccat ttgtctccac acctccgctt acatcaacac caataacgcc
     6301 atttaatcta agcgcatcac caacattttc tggcgtcagt ccaccagcta acataaaatg
     6361 taagctttcg gggctctctt gccttccaac ccagtcagaa atcgagttcc aatccaaaag
     6421 ttcacctgtc ccacctgctt ctgaatcaaa caagggaata aacgaatgag gtttctgtga
     6481 agctgcactg agtagtatgt tgcagtcttt tggaaatacg agtcttttaa taactggcaa
     6541 accgaggaac tcttggtatt cttgccacga ctcatctcca tgcagttgga cgatatcaat
     6601 gccgtaatca ttgaccagag ccaaaacatc ctccttaggt tgattacgaa acacgccaac
     6661 caagtatttc ggagtgcctg aactattttt atatgctttt acaagacttg aaattttcct
     6721 tgcaataacc gggtcaattg ttctctttct attgggcaca catataatac ccagcaagtc
     6781 agcatcggaa tctagagcac attctgcggc ctctgtgctc
//
