LOCUS       sequence-142655-        6245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Centromeric plasmid to construct episomal C-terminal fusions of 
            genes with yeast-adapted TagRFP-T and URA3 selection marker .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6245)
  AUTHORS   Malcova I, Farkasovsky M, Senohrabkova L, Vasicova P, Hasek J
  TITLE     New integrative modules for multicolor-protein labeling and 
            live-cell imaging in Saccharomyces cerevisiae.
  JOURNAL   FEMS Yeast Res. 2016 Mar 17. pii: fow027.
  PUBMED    26994102
REFERENCE   2  (bases 1 to 6245)
  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: "FEMS Yeast 
            Res. 2016 Mar 17. pii: fow027."
COMMENT     Sequence Label: pIM35
FEATURES             Location/Qualifiers
     source          1..6245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        196..416
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             417..1220
                     /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"
     rep_origin      complement(1351..1806)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1488..1509)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1700..1719
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1936..1958
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1950..1967
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1951..1967
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1977..1996
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1977..1995
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(2014..2261)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     2227..2245
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3054..3070)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3054..3070)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(3071..3420)
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     complement(3471..3491)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3471..3489)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3510..3526)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3510..3526)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3523..3545)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3534..3550
                     /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)."
     promoter        complement(join(3558..3564,3565..3582,3583..3588))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3741..3758)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3912..4500)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3992..4011)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4671..5462,5463..5531))
                     /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     5294..5313
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5532..5636)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5673..6176
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6218..6236
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
      241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
      961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
     1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
     1981 acgactcact atagggcgaa ttgggtaccg gccgcaaatt aaagccttcg agcgtcccaa
     2041 aaccttctca agcaaggttt tcagtataat gttacatgcg tacacgcgtc tgtacagaaa
     2101 aaaaagaaaa atttgaaata taaataacgt tcttaatact aacataacta taaaaaaata
     2161 aatagggacc tagacttcag gttgtctaac tccttccttt tcggttagag cggatgtggg
     2221 gggagggcgt gaatgtaagc gtgacataac taattacatg actcgaccag ttttacttat
     2281 acaattcgtc cataccgttc aacttatgac ctaacttgga aggcaaatcg cagtatcttg
     2341 caacagcgac ttcgtgttgt tcaacatatg tttccttgtc tgcttcttta attctttcca
     2401 atctatgatc gacgtagtat acaccaggca tcttcaagtt tttagctggc tttttagatc
     2461 tgtaagtggt cttgaaattg caaattaagt gaccaccacc aactaatttc aatgccatgt
     2521 cagttcgacc ttctaaacca ccatcggcag gatacaacat ttcggtgttt gcttcccaac
     2581 ccaaagtttt cttttgcatt acaggaccgt tacttgggaa gtttacacct ctgatcttaa
     2641 cgttatagat taaacaaccg tcttgcaagg atgtatcttg tgtggcagtc aaaacaccac
     2701 cgtcttcata tgtagtgact ctttcccaag tgaaaccttc agggaatgat tgcttaaaga
     2761 aatctggtat accttgagta tgattgatga aggttcttga accgtacata aaagatgtag
     2821 ctaagatatc gaaggcaaaa ggcaatggac caccttctac aactttaatt ctcatggttt
     2881 gtgtaccttc atatggttta ccttcacctt cagaggtaca cttaaaatgg tggttattta
     2941 cagtaccttc catatacaac ttcatgtgca tgttttcctt aatcaattct tcacccttag
     3001 ataccatgtc gacggtatcg ataagcttga tatcgaattc ctgcagcccg ggggatccac
     3061 tagttctaga gtatggatgg gggtaataga attgtatcta tgtatctgac gaccctgtat
     3121 tacacgaagg aaatagtaga tgaaaagaca agagagcaag aaaaaggaaa aacttccttt
     3181 ctaacagacg ctttacttaa ccttatatat atcttatttt tttcatcgag cgtgttcaat
     3241 tggacaaggt gccacctttt cgacacttcg gttattatgt tacacagttt tcatgaggat
     3301 ggcgccttga ctaacttaat tagtcatttg ccaaccacca cagttcccca atatcgacag
     3361 cttcacgtgc catttgccat tttgcgatca tgtgacctca agagaaaaag caaaaaataa
     3421 tgagagctaa gggattcgaa cccttgcatc cggagctcca gcttttgttc cctttagtga
     3481 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3541 ccgctcacaa ttccacacaa cataggagcc ggaagcataa agtgtaaagc ctggggtgcc
     3601 taatgagtga ggtaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3661 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3721 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3781 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3841 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3901 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3961 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4021 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4081 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4141 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4201 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4261 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4321 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     4381 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4441 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4501 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4561 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4621 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4681 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4741 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4801 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4861 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4921 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4981 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5041 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5101 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5161 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5221 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5281 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5341 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5401 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5461 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5521 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5581 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5641 tttccccgaa aagtgccacc tgggtccttt tcatcacgtg ctataaaaat aattataatt
     5701 taaatttttt aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa
     5761 aaaatagttt ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta
     5821 ccttttatct tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt
     5881 gtagaagacc acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt
     5941 atatctattt aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt
     6001 gaaatttttt aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt
     6061 tatttacttt ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc
     6121 caaattattc catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc
     6181 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     6241 tcgtc
//
