LOCUS       sequence-332095-        7763 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses Lifeact-mScarletI 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  TITLE     Teusink unpublished plasmids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 Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: LifeAct-mScarletI pDRF1-GW
FEATURES             Location/Qualifiers
     source          1..7763
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    232..256
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             305..355
                     /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"
     regulatory      371..380
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             377..1072
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(510..528)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     778..797
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1085..1109)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1171..1190)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1172..1190)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      1338..1525
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1393..1417)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(1537..1554)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1537..1553)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1546..1568)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1989..3335
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(3446..4249)
                     /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(4250..4465)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4490..4508)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4576..4680
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4681..4749,4750..5541)
                     /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(4899..4918)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5712..6300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6201..6220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6454..6471
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6588..6609
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6624..6629,6630..6647,6648..6654)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6662..6678
                     /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     6667..6689
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6686..6702
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6686..6702
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      complement(6732..7187)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6869..6890)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7081..7100
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tctcttaaat tttttctctt tctttcctat aacaccaata gtgaaaatct ttttttcttc
       61 tatatctaca aaaacttttt ttttctatca acctcgttga taaatttttt ctttaacaat
      121 cgttaataat taattaattg gaaaataacc attttttctc tcttttatac acacattcaa
      181 aagaaagaaa aaaaatatac cccagcctcg actagtgaat tcgcggccat cacaagtttg
      241 tacaaaaaag caggctgcta gcgctaccgg actcagatct cgagctcaag cttcgaattc
      301 caccatgggc gtggccgacc tgatcaagaa gttcgagagc atcagcaagg aggaggggga
      361 tccaccggtc gccaccatgg tgagcaaggg cgaggcagtg atcaaggagt tcatgcggtt
      421 caaggtgcac atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga
      481 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
      541 gcccttctcc tgggacatcc tgtcccctca gttcatgtac ggctccaggg ccttcatcaa
      601 gcaccccgcc gacatccccg actactataa gcagtccttc cccgagggct tcaagtggga
      661 gcgcgtgatg aacttcgagg acggcggcgc cgtgaccgtg acccaggaca cctccctgga
      721 ggacggcacc ctgatctaca aggtgaagct ccgcggcacc aacttccctc ctgacggccc
      781 cgtaatgcag aagaagacaa tgggctggga agcgtccacc gagcggttgt accccgagga
      841 cggcgtgctg aagggcgaca ttaagatggc cctgcgcctg aaggacggcg gccgctacct
      901 ggcggacttc aagaccacct acaaggccaa gaagcccgtg cagatgcccg gcgcctacaa
      961 cgtcgaccgc aagttggaca tcacctccca caacgaggac tacaccgtgg tggaacagta
     1021 cgaacgctcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaagcgg
     1081 ccgcacccag ctttcttgta caaagtggtg tagttaattc atgatggccg ctgcaggtcg
     1141 acctcgaggg ggggcccggt acccaattcg ccctatagtg agtcgtatta cgcgcggatc
     1201 cagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt gtcggcttgt
     1261 ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt agatacgttg
     1321 ttgacacttc taaataagcg aatttcttat gatttatgat ttttattatt aaataagtta
     1381 taaaaaaaat aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct
     1441 tattcttgag taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg
     1501 ctcttattga ccacacctct accggcatgc caattcactg gccgtcgttt tacaacgtcg
     1561 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     1621 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     1681 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     1741 ccgcataatc ggatcgtact tgttacccat cattgaattt tgaacatccg aacctgggag
     1801 ttttccctga aacagatagt atatttgaac ctgtataata atatatagtc tagcgcttta
     1861 cggaagacaa tgtatgtatt tcggttcctg gagaaactat tgcatctatt gcataggtaa
     1921 tcttgcacgt cgcatccccg gttcattttc tgcgtttcca tcttgcactt caatagcata
     1981 tctttgttaa cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc
     2041 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag
     2101 cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag
     2161 agcgctaatt tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc
     2221 gagagcgcta ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc
     2281 ccgagagcgc tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct
     2341 ctataatgca gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc
     2401 tactttggtg tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact
     2461 gattactagc gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct
     2521 ataccgatgt ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca
     2581 ttggtcagaa aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa
     2641 tgtttacatt ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt
     2701 tgtctaaaga gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa
     2761 gttcaaggag cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag
     2821 caaagagata cttttgagca atgtttgtgg aagcggtatt cgcaatattt tagtagctcg
     2881 ttacagtccg gtgcgttttt ggttttttga aagtgcgtct tcagagcgct tttggttttc
     2941 aaaagcgctc tgaagttcct atactttcta gctagagaat aggaacttcg gaataggaac
     3001 ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa cgcgagctgc gcacatacag
     3061 ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt atatatatat acatgagaag
     3121 aacggcatag tgcgtgttta tgcttaaatg cgtacttata tgcgtctatt tatgtaggat
     3181 gaaaggtagt ctagtacctc ctgtgatatt atcccattcc atgcggggta tcgtatgctt
     3241 ccttcagcac taccctttag ctgttctata tgctgccact cctcaattgg attagtctca
     3301 tccttcaatg ctatcatttc ctttgatatt ggatcgatcc gatgataagc tgtcaaacat
     3361 gagaattggg taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac
     3421 atgcatttac ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt
     3481 cccagcctgc ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata
     3541 gtcctcttcc aacaataata atgtcagatc ctgtagagac cacatcatcc acggttctat
     3601 actgttgacc caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc
     3661 aatcgtaacc ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat
     3721 ctttgtcgct cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc
     3781 ccttgcatga caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg
     3841 ccgcctgctt caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt
     3901 ctgcccattc tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa
     3961 tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta
     4021 gcggcttaac tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta
     4081 aacaaatttt gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat
     4141 ccaatgaagc acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa
     4201 caggactagg atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc
     4261 gtttcctgca tgtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt
     4321 cttcaacact acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt
     4381 ccttctgttc ggagattacc gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag
     4441 aataaaaaaa aaatgatgaa ttgaaaagct aattcttgaa gacgaaaggg cctcgtgata
     4501 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     4561 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4621 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4681 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4741 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4801 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4861 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4921 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4981 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5041 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5101 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5161 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5221 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5281 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5341 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5401 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5461 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5521 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5581 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5641 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5701 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5761 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5821 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5881 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5941 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6001 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6061 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6121 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6181 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6241 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6301 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6361 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     6421 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     6481 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     6541 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     6601 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     6661 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt
     6721 accgcatcag gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     6781 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     6841 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     6901 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     6961 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     7021 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     7081 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     7141 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccaagc
     7201 ttcctgaaac ggagaaacat aaacaggcat tgctgggatc acccatacat cactctgttt
     7261 tgcctgacct tttccggtaa tttgaaaaca aacccggtct cgaagcggag atccggcgat
     7321 aattaccgca gaaataaacc catacacgag acgtagaacc agccgcacat ggccggagaa
     7381 actcctgcga gaatttcgta aactcgcgcg cattgcatct gtatttccta atgcggcact
     7441 tccaggcctc gatcgagacc gtttatccat tgcttttttg ttgtcttttt ccctcgttca
     7501 cagaaagtct gaagaagcta tagtagaact atgagctttt tttgtttctg ttttcctttt
     7561 tttttttttt acctctgtgg aaattgttac tctcacactc tttagttcgt ttgtttgttt
     7621 tgtttattcc aattatgacc ggtgacgaaa cgtggtcgat ggtgggtacc gcttatgctc
     7681 ccctccatta gtttcgatta tataaaaagg ccaaatattg tattattttc aaatgtccta
     7741 tcattatcgt ctaacatcta att
//
