LOCUS       sequence-199926-        8496 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for pop-in/pop-out replacement of Pik1 with iGFP-Pik1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8496)
  AUTHORS   Day KJ, Casler JC, Glick BS
  TITLE     Budding Yeast Has a Minimal Endomembrane System.
  JOURNAL   Dev Cell. 2018 Jan 8;44(1):56-72.e4. doi: 
            10.1016/j.devcel.2017.12.014. Epub 2018 Jan 8.
  PUBMED    29316441
REFERENCE   2  (bases 1 to 8496)
  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"; doi: 
            "10.1016/j.devcel.2017.12.014"; journalName: "Dev Cell"; date: 
            "2018-01-8- 8"; volume: "44"; issue: "1"; pages: "56-72.e4"
COMMENT     Sequence Label: YIplac211-iGFP-PIK1
FEATURES             Location/Qualifiers
     source          1..8496
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(753..781)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(4989..5006)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4989..5005)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4998..5020)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5214..5233)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5333..5355
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5473..6276)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6277..6497)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6504..6522)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6590..6694
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6695..6763,6764..7555)
                     /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(6913..6932)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7726..8314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8215..8234
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8468..8485
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
      241 atgccttcaa aatagaaaaa aataaaaaaa tagacgtttc attaatcaaa atagcctgta
      301 gagattttga gggattgccc caaactttat gtataatatc accacttatt attcactatt
      361 catagtatgt tacatcataa ggccattgtc accttcggcc agcgatagta ctcaaatcta
      421 taataaattt tccatattat cctttacgta cttgatagcg tgtataacag ggtcattatt
      481 cacgatatgt tcaactgttg tcattttcgt gttattgcac tgaaacaaaa gaaccctacc
      541 aaaaaattcg ttagagaaaa actaaaagta caagatctta tactgaagac ccttcatttt
      601 ttaggcccaa cttctatatt aagaagatcc ctggaccctg ctagaaattt tcaattagct
      661 tatcattttt cttcattgcg agcgttcgtt ttcattaagc attcgttctc ttcactatgc
      721 atatggatag cactgagagc cttttcactg gagttgtccc aattcttgtt gaattagatg
      781 gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg
      841 gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcct tggccaacac
      901 ttgtcactac tttaacttat ggtgttcagt gcttttcaag atacccggat catatgaaac
      961 agcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt
     1021 tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg
     1081 ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttgggcaca
     1141 aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg
     1201 gaatcaaagc taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag
     1261 accattatca acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt
     1321 acctgtccac acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc
     1381 ttaaagagtt tgtaacagct gctgggatta cacatggcag ctccggctcc agcgggaaag
     1441 catccagttc aaagaaaagc tttgatgata ctattgaact aaaaaaaaat gaacaattac
     1501 tgaaactgat caactctagt gaattcaccc ttcacaattg tgtggagtta ctgtgcaaac
     1561 actccgaaaa tattggtata cattactatt tatgtcagaa attggccaca tttcctcaca
     1621 gcgaactaca gttctatatt ccccaactag tacaggtcct cgtcaccatg gagacagaat
     1681 caatggcttt agaagattta ctattaaggt tgagggcgga aaaccctcat tttgcactac
     1741 tgacgttttg gcagttacaa gcgttactaa cggatttatc tactgacccc gcttcttatg
     1801 gcttccaagt ggctagaaga gtcttaaaca acttacaaac caatctcttt aacacaagtt
     1861 caggtagcga taaaaatgtt aaaatacatg aaaacgttgc accggcctta gttctttcct
     1921 ctatgataat gtcggctata gcattccccc aattaagcga agtgaccaaa ccattagtgg
     1981 aatctcaagg tagaagacaa aaagctttcg tttttaagtt ggctagaagt gcaatgaaag
     2041 attttaccaa gaacatgacc ttgaaaaata ccctactaaa caagaaaact tccagatcca
     2101 aaagagttag ctcaaatcgc agttcaactc cgacttctcc aatagattta atagatccaa
     2161 taaaaactaa agaagatgca tccttcagga aatccagaca tagtgaggta aaattggatt
     2221 tcgacattgt ggatgatata ggtaaccagg tctttgaaga aagaatctca tcttcaatca
     2281 aactcccaaa acgtaaacct aagtatttgg ataattcata cgttcatagg acatatgatg
     2341 gcaaaaatat aaacagggac ggaagcattt caaataccgc aaaggctctc gatggaaata
     2401 aaggtgatta tatttctcca aagggacgta atgatgaaaa taatgagatt ggtaacaatg
     2461 aagatgaaac tggtggtgaa acggaggagg acgcggacgc tttaaactct gatcacttca
     2521 ccagttctat gccagatctg cataatattc aaccaaggac ttcttctgct tcatctgctt
     2581 ctttagaagg gacacctaag ttaaacagga ccaattccca acccctttcg cgccaggcat
     2641 tcaaaaacag taaaaaagcg aattcttctt tgagtcaaga aattgacttg tcgcaattat
     2701 cgaccacttc aaaaataaaa atgttaaaag caaattattt tcgttgcgag acacaatttg
     2761 ccattgcgct agaaacaata tctcaaaggc tagctcgagt accgacagaa gctagattga
     2821 gtgccttacg agccgagttg tttctattaa acagggacct accagcggaa gtagatatcc
     2881 ccactttatt gcctccaaat aaaaaaggaa agttacataa attagtgacc attactgcta
     2941 atgaggcaca agttttgaac tctgcagaaa aagttccata tttactcctg atagaatact
     3001 tgagagacga atttgatttt gacccaacaa gtgaaaccaa tgaaagatta ttaaagaaga
     3061 tcagtggtaa tcagggaggc ttgatatttg atttaaatta catgaataga aaagaaaaca
     3121 acgaaaatag aaatgaaagt actcttacta gcaacaacac tcgatcttcg gtatatgata
     3181 gtaactcgtt caacaacggg gcctcccgca atgaaggtct atccagtacc tcaagaagtg
     3241 attcagcgtc cacagctcat gttaggactg aagtcaataa agaggaagat ttaggtgata
     3301 tgtcaatggt aaaagtcagg aacagaacgg atgacgaagc gtatagaaat gctttagtaa
     3361 tacagagtgc cgcaaatgtt ccaattttac ctgatgatag tcaagacaga agcccagagt
     3421 tgaactttgg ctcaaacttg gacgaagtac tcatcgagaa tggaattaat agcaaaaata
     3481 tacatagcca aactgacgct ttagcagacc agatgagagt ttcagctgtt atgttagcac
     3541 aattggataa gtcgccacaa cagttatcag aaagtaccaa acaaatccgc gctcaaatta
     3601 tttcatcaat gaaggaggtg caggataaat ttggttacca tgatttggaa gcccttcatg
     3661 gaatggcagg tgagagaaaa ttggaaaacg atttaatgac gggtggtatt gacacttcat
     3721 atctaggtga agattgggct accaagaaag agaggatacg taaaacttcg gaatatggcc
     3781 atttcgaaaa ctgggattta tgttctgtaa tcgccaagac gggtgatgat ttgagacagg
     3841 aggcgtttgc ataccagatg attcaagcga tggccaatat ttgggttaaa gaaaaagttg
     3901 acgtttgggt taaaagaatg aaaattttaa ttactagtgc gaatacggga cttgtggaga
     3961 ccatcacaaa tgctatgtct gtgcatagta ttaaaaaggc tttaaccaaa aaaatgattg
     4021 aagatgcaga actagatgac aagggtggta ttgcctcttt gaatgatcac ttccttagag
     4081 cttttggtaa tcctaatgga tttaagtata gaagagcaca agacaacttt gcttcttcgt
     4141 tagccgcata ttctgtcatt tgctatctct tgcaggttaa agatagacac aacggtaaca
     4201 ttatgatcga taacgaaggc catgtaagtc acatcgattt tggatttatg ctatcaaatt
     4261 cacccggctc agtgggcttt gaggccgcac cattcaaatt aacttacgaa tatattgaac
     4321 tgctaggcgg agtagaggga gaagcgttta aaaagtttgt tgaactaact aaaagttcgt
     4381 tcaaggctct gagaaagtat gctgatcaaa tcgtatcaat gtgtgagatt atgcaaaagg
     4441 acaatatgca gccttgtttc gatgctggcg aacaaacaag tgtacaacta cgacaaaggt
     4501 tccaattgga cttatcagaa aaagaagttg atgacttcgt agaaaatttc ttgataggta
     4561 aatctttggg tagtatttac accagaatat acgaccaatt tcaacttatt acacagggta
     4621 tatatagctg agaaggaaaa tagctgtgac agaactggaa aaacaatttg caaatgtccg
     4681 gagaacaaaa aaattggaat tttagtaaaa ggaatataca tatatttatt tattgccgag
     4741 aataatagag cagctgactc agaaaaagca aggtaaaaga atgagagtaa aataaatatg
     4801 tggttaatta accatacata taaattatta tcttattttc tgcgtaaagc gtcaaaccgt
     4861 ttcttcaact catctagttc atcattttct tgctccttct tagtctttct ttttgtagtt
     4921 ttcttttctg attgtttctt ttcgatcact tcctttttga tatctttcgg aatcttgagc
     4981 tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     5041 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     5101 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat
     5161 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     5221 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     5281 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5341 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcgggtaa
     5401 taactgatat aattaaattg aagctctaat ttgtgagttt agtatacatg catttactta
     5461 taatacagtt ttttagtttt gctggccgca tcttctcaaa tatgcttccc agcctgcttt
     5521 tctgtaacgt tcaccctcta ccttagcatc ccttcccttt gcaaatagtc ctcttccaac
     5581 aataataatg tcagatcctg tagagaccac atcatccacg gttctatact gttgacccaa
     5641 tgcgtctccc ttgtcatcta aacccacacc gggtgtcata atcaaccaat cgtaaccttc
     5701 atctcttcca cccatgtctc tttgagcaat aaagccgata acaaaatctt tgtcgctctt
     5761 cgcaatgtca acagtaccct tagtatattc tccagtagat agggagccct tgcatgacaa
     5821 ttctgctaac atcaaaaggc ctctaggttc ctttgttact tcttctgccg cctgcttcaa
     5881 accgctaaca atacctgggc ccaccacacc gtgtgcattc gtaatgtctg cccattctgc
     5941 tattctgtat acacccgcag agtactgcaa tttgactgta ttaccaatgt cagcaaattt
     6001 tctgtcttcg aagagtaaaa aattgtactt ggcggataat gcctttagcg gcttaactgt
     6061 gccctccatg gaaaaatcag tcaagatatc cacatgtgtt tttagtaaac aaattttggg
     6121 acctaatgct tcaactaact ccagtaattc cttggtggta cgaacatcca atgaagcaca
     6181 caagtttgtt tgcttttcgt gcatgatatt aaatagcttg gcagcaacag gactaggatg
     6241 agtagcagca cgttccttat atgtagcttt cgacatgatt tatcttcgtt tcctgcatgt
     6301 ttttgttctg tgcagttggg ttaagaatac tgggcaattt catgtttctt caacactaca
     6361 tatgcgtata tataccaatc taagtctgtg ctccttcctt cgttcttcct tctgttcgga
     6421 gattaccgaa tcaaaaaaat ttcaaagaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa
     6481 tgatgaattg aattgaaaag ctggcctcgt gatacgccta tttttatagg ttaatgtcat
     6541 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6601 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6661 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6721 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6781 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     6841 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     6901 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6961 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     7021 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     7081 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     7141 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7201 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7261 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7321 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     7381 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     7441 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7501 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7561 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7621 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7681 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     7741 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     7801 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     7861 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     7921 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     7981 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     8041 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     8101 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     8161 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     8221 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     8281 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     8341 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     8401 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     8461 cgagcgcagc gagtcagtga gcgaggaagc ggaaga
//
