LOCUS       sequence-332145-        7763 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Lifeact-mScarlet to visualize actin in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7763)
  TITLE     Teusink unpublished plasmids
REFERENCE   2  (bases 1 to 7763)
  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"
COMMENT     Sequence Label: LifeAct-mScarlet pDRF1-GW
FEATURES             Location/Qualifiers
     source          1..7763
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..52)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(206..794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(286..305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(965..1756,1757..1825))
                     /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     1588..1607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1826..1930)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1998..2016
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2041..2256
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2257..3060
                     /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      3171..4517
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     4938..4960
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4952..4969
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4953..4969
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(4981..5168)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5089..5113
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     5316..5335
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5316..5334
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5397..5421
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(5434..6129)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(5709..5728)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5978..5996
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      6126..6135
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(6151..6201)
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     protein_bind    complement(6250..6274)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     rep_origin      7082..7537
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7169..7188)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7379..7400
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7567..7583)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7567..7583)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7580..7602)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7591..7607
                     /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(7615..7621,7622..7639,7640..7645))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7660..7681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
       61 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      121 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      181 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      241 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      301 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      361 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      421 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      481 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      541 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      601 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      661 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      721 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      781 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      841 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
      901 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
      961 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1021 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1081 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     1141 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1201 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1261 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1321 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1381 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1441 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1501 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1561 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1621 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1681 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     1741 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     1801 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     1861 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1921 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     1981 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattagctt
     2041 ttcaattcat catttttttt ttattctttt ttttgatttc ggtttccttg aaattttttt
     2101 gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg agcacagact tagattggta
     2161 tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg
     2221 cacagaacaa aaacatgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa
     2281 cgtgctgcta ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag
     2341 caaacaaact tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt
     2401 gaagcattag gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt
     2461 tccatggagg gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc
     2521 ttcgaagaca gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt
     2581 gtatacagaa tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt
     2641 attgttagcg gtttgaagca ggcggcagaa gaagtaacaa aggaacctag aggccttttg
     2701 atgttagcag aattgtcatg caagggctcc ctatctactg gagaatatac taagggtact
     2761 gttgacattg cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg
     2821 ggtggaagag atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac
     2881 aagggagacg cattgggtca acagtataga accgtggatg atgtggtctc tacaggatct
     2941 gacattatta ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt
     3001 gaacgttaca gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa
     3061 aaaactgtat tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa
     3121 ttatatcagt tattacccaa ttctcatgtt tgacagctta tcatcggatc gatccaatat
     3181 caaaggaaat gatagcattg aaggatgaga ctaatccaat tgaggagtgg cagcatatag
     3241 aacagctaaa gggtagtgct gaaggaagca tacgataccc cgcatggaat gggataatat
     3301 cacaggaggt actagactac ctttcatcct acataaatag acgcatataa gtacgcattt
     3361 aagcataaac acgcactatg ccgttcttct catgtatata tatatacagg caacacgcag
     3421 atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc tcgcgttgca ttttcggaag
     3481 cgctcgtttt cggaaacgct ttgaagttcc tattccgaag ttcctattct ctagctagaa
     3541 agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc tctgaagacg cactttcaaa
     3601 aaaccaaaaa cgcaccggac tgtaacgagc tactaaaata ttgcgaatac cgcttccaca
     3661 aacattgctc aaaagtatct ctttgctata tatctctgtg ctatatccct atataaccta
     3721 cccatccacc tttcgctcct tgaacttgca tctaaactcg acctctacat tttttatgtt
     3781 tatctctagt attactcttt agacaaaaaa attgtagtaa gaactattca tagagtgaat
     3841 cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt atatagagac aaaatagaag
     3901 aaaccgttca taattttctg accaatgaag aatcatcaac gctatcactt tctgttcaca
     3961 aagtatgcgc aatccacatc ggtatagaat ataatcgggg atgcctttat cttgaaaaaa
     4021 tgcacccgca gcttcgctag taatcagtaa acgcgggaag tggagtcagg ctttttttat
     4081 ggaagagaaa atagacacca aagtagcctt cttctaacct taacggacct acagtgcaaa
     4141 aagttatcaa gagactgcat tatagagcgc acaaaggaga aaaaaagtaa tctaagatgc
     4201 tttgttagaa aaatagcgct ctcgggatgc atttttgtag aacaaaaaag aagtatagat
     4261 tctttgttgg taaaatagcg ctctcgcgtt gcatttctgt tctgtaaaaa tgcagctcag
     4321 attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttttacaa aaatgaagca
     4381 cagattcttc gttggtaaaa tagcgctttc gcgttgcatt tctgttctgt aaaaatgcag
     4441 ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttc tacaaaatga
     4501 agcacagatg cttcgttaac aaagatatgc tattgaagtg caagatggaa acgcagaaaa
     4561 tgaaccgggg atgcgacgtg caagattacc tatgcaatag atgcaatagt ttctccagga
     4621 accgaaatac atacattgtc ttccgtaaag cgctagacta tatattatta tacaggttca
     4681 aatatactat ctgtttcagg gaaaactccc aggttcggat gttcaaaatt caatgatggg
     4741 taacaagtac gatccgatta tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     4801 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     4861 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     4921 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattggcatg
     4981 ccggtagagg tgtggtcaat aagagcgacc tcatgctata cctgagaaag caacctgacc
     5041 tacaggaaag agttactcaa gaataagaat tttcgtttta aaacctaaga gtcactttaa
     5101 aatttgtata cacttatttt ttttataact tatttaataa taaaaatcat aaatcataag
     5161 aaattcgctt atttagaagt gtcaacaacg tatctaccaa cgatttgacc cttttccatc
     5221 ttttcgtaaa tttctggcaa ggtagacaag ccgacaacct tgattggaga cttgaccaaa
     5281 cctctggcga agaagtccaa agctggatcc gcgcgtaata cgactcacta tagggcgaat
     5341 tgggtaccgg gccccccctc gaggtcgacc tgcagcggcc atcatgaatt aactacacca
     5401 ctttgtacaa gaaagctggg tgcggccgct ttacttgtac agctcgtcca tgccgccggt
     5461 ggagtggcgg ccctcggagc gttcgtactg ttccaccacg gtgtagtcct cgttgtggga
     5521 ggtgatgtcc aacttgcggt cgacgttgta ggcgccgggc atctgcacgg gcttcttggc
     5581 cttgtaggtg gtcttgaagt ccgccaggta gcggccgccg tccttcaggc gcagggccat
     5641 cttaatgtcg cccttcagca cgccgtcctc ggggtacaac cgctcggtgg acgcttccca
     5701 gcccattgtc ttcttctgca ttacggggcc gtcaggaggg aagttggtgc cgcggagctt
     5761 caccttgtag atcagggtgc cgtcctccag ggaggtgtcc tgggtcacgg tcacggcgcc
     5821 gccgtcctcg aagttcatca cgcgctccca cttgaagccc tcggggaagg actgcttata
     5881 gtagtcgggg atgtcggcgg ggtgcttggt gaaggccctg gagccgtaca tgaactgagg
     5941 ggacaggatg tcccaggaga agggcagggg gccacccttg gtcaccttca gcttggcggt
     6001 ctgggtgccc tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt
     6061 catggagccc tccatgtgca ccttgaaccg catgaactcc ttgatcactg cctcgccctt
     6121 gctcaccatg gtggcgaccg gtggatcccc ctcctccttg ctgatgctct cgaacttctt
     6181 gatcaggtcg gccacgccca tggtggaatt cgaagcttga gctcgagatc tgagtccggt
     6241 agcgctagca gcctgctttt ttgtacaaac ttgtgatggc cgcgaattca ctagtcgagg
     6301 ctggggtata ttttttttct ttcttttgaa tgtgtgtata aaagagagaa aaaatggtta
     6361 ttttccaatt aattaattat taacgattgt taaagaaaaa atttatcaac gaggttgata
     6421 gaaaaaaaaa gtttttgtag atatagaaga aaaaaagatt ttcactattg gtgttatagg
     6481 aaagaaagag aaaaaattta agagaaatta gatgttagac gataatgata ggacatttga
     6541 aaataataca atatttggcc tttttatata atcgaaacta atggagggga gcataagcgg
     6601 tacccaccat cgaccacgtt tcgtcaccgg tcataattgg aataaacaaa acaaacaaac
     6661 gaactaaaga gtgtgagagt aacaatttcc acagaggtaa aaaaaaaaaa aaggaaaaca
     6721 gaaacaaaaa aagctcatag ttctactata gcttcttcag actttctgtg aacgagggaa
     6781 aaagacaaca aaaaagcaat ggataaacgg tctcgatcga ggcctggaag tgccgcatta
     6841 ggaaatacag atgcaatgcg cgcgagttta cgaaattctc gcaggagttt ctccggccat
     6901 gtgcggctgg ttctacgtct cgtgtatggg tttatttctg cggtaattat cgccggatct
     6961 ccgcttcgag accgggtttg ttttcaaatt accggaaaag gtcaggcaaa acagagtgat
     7021 gtatgggtga tcccagcaat gcctgtttat gtttctccgt ttcaggaagc ttggcgaatg
     7081 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     7141 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     7201 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     7261 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     7321 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     7381 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     7441 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     7501 aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtaa gcttggcgta
     7561 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     7621 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     7681 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7741 atgaatcggc caacgcgcgg gga
//
