LOCUS       sequence-469859-        7372 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration vector for expressing Lifeact-3xmNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7372)
  AUTHORS   Wirshing ACE, Goode BL
  TITLE     Improved tools for live imaging of F-actin structures in yeast.
  JOURNAL   Mol Biol Cell. 2024 Sep 1;35(9):mr7. doi: 10.1091/mbc.E24-05-0212-T.
            Epub 2024 Jul 18.
  PUBMED    39024291
REFERENCE   2  (bases 1 to 7372)
  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: "Mol Biol 
            Cell."; date: "2024-09-1"; volume: "35(9):mr7. doi"; pages: " 
            10.1091/mbc.E24-05-0212-T. Epub 2024 Jul 18"
COMMENT     Sequence Label: Lifeact-3xmNG::TRP1
FEATURES             Location/Qualifiers
     source          1..7372
                     /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        191..472
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             473..1147
                     /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"
     rep_origin      complement(1250..1705)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1387..1408)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1599..1618
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1831..1853
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1845..1862
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1846..1862
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1869..1888
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1869..1887
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1920..1936
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1920..1936
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             2585..2635
                     /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"
     CDS             2660..3367
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2915..2924
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3386..4093
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      3641..3650
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4112..4819
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      4367..4376
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     terminator      4852..5039
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4907..4931)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5067..5083)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5068..5084)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(5112..5130)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5151..5167)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5151..5167)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5164..5186)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5175..5191
                     /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(5199..5205,5206..5223,5224..5229))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5244..5265
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5382..5399)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5553..6141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5633..5652)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6312..7103,7104..7172))
                     /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     6935..6954
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7173..7277)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7345..7363
                     /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 accatagatc aacgacatta ctatatatat aatataggaa gcatttaata gaacagcatc
      241 gtaatatatg tgtactttgc agttatgacg ccagatggca gtagtggaag atattcttta
      301 ttgaaaaata gcttgtcacc ttacgtacaa tcttgatccg gagcttttct ttttttgccg
      361 attaagaatt aattcggtcg aaaaaagaaa aggagagggc caagagggag ggcattggtg
      421 actattgagc acgtgagtat acgtgattaa gcacacaaag gcagcttgga gtatgtctgt
      481 tattaatttc acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga
      541 ggccgcagaa tgtgctctag attccgatgc tgacttgctg ggtattatat gtgtgcccaa
      601 tagaaagaga acaattgacc cggttattgc aaggaaaatt tcaagtcttg taaaagcata
      661 taaaaatagt tcaggcactc cgaaatactt ggttggcgtg tttcgtaatc aacctaagga
      721 ggatgttttg gctctggtca atgattacgg cattgatatc gtccaactgc atggagatga
      781 gtcgtggcaa gaataccaag agttcctcgg tttgccagtt attaaaagac tcgtatttcc
      841 aaaagactgc aacatactac tcagtgcagc ttcacagaaa cctcattcgt ttattccctt
      901 gtttgattca gaagcaggtg ggacaggtga acttttggat tggaactcga tttctgactg
      961 ggttggaagg caagagagcc ccgaaagctt acattttatg ttagctggtg gactgacgcc
     1021 agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt ggtgttgatg taagcggagg
     1081 tgtggagaca aatggtgtaa aagactctaa caaaatagca aatttcgtca aaaatgctaa
     1141 gaaataggtt attactgagt agtatttatt taagtattgt ttgtgcactt gccgatctat
     1201 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     1261 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     1321 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     1381 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     1441 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     1501 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     1561 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     1621 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     1681 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     1741 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     1801 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     1861 gtgaattgta atacgactca ctatagggcg aattggagct ccaccgcggt ggcggccgct
     1921 ctagaactag tggatcccgg gtttgcgttt aagatgccaa gattcatcag ctgtcatgcc
     1981 aaggccattg attacccaca atcaaacata acctacatag gaattgaccc taaaccagca
     2041 aactacaacc aaacacaact ttcaaaatac tatgatgatc tagtacaaat ggaagataaa
     2101 aatgcactaa gcttattttc ttcagattgg tacgctacaa aggatagatt actcaccaag
     2161 aaaagatctc gcaacccatt taacagaact gcaccgtacg ctcagaatat tttctgcaaa
     2221 gaaaacggca agaggatcga gggaatcgag gatgacgagg aatattttga aacaaaaatt
     2281 aagtgtaaaa tgccatggtc gtcgccaaga caataaacgt gtgggagtat tgcaaaaaca
     2341 tagtatataa gcgtaaatca ctatataaag cccaaagaag ttagcactaa agcatcattc
     2401 atcgttcaaa actcctctct caagtgagag aattcggcgc tgaaaaaaag ctttgtcaaa
     2461 accaagaaaa aaacgtagga ttggacacga ggttcttttt ttaatgtaaa caagaagtgg
     2521 cactatagct gtgaaacttc caatacaatt gtataccaaa taagctatta caaattgatt
     2581 taaaatgggt gtcgcagatt tgatcaagaa attcgaaagc atctcaaagg aagaaggtga
     2641 cggtgctggt ttaattaaca tggtgagcaa gggcgaggag gataacatgg cctctctccc
     2701 agcgacacat gagttacaca tctttggctc catcaacggt gtggactttg acatggtggg
     2761 tcagggcacc ggcaatccaa atgatggtta tgaggagtta aacctgaagt ccaccaaggg
     2821 tgacctccag ttctccccct ggattctggt ccctcatatc gggtatggct tccatcagta
     2881 cctgccctac cctgacggga tgtcgccttt ccaggccgcc atggtagatg gctccggcta
     2941 ccaagtccat cgcacaatgc agtttgaaga tggtgcctcc cttactgtta actaccgcta
     3001 cacctacgag ggaagccaca tcaaaggaga ggcccaggtg aaggggactg gtttccctgc
     3061 tgacggtcct gtgatgacca actcgctgac cgctgcggac tggtgcaggt cgaagaagac
     3121 ttaccccaac gacaaaacca tcatcagtac ctttaagtgg agttacacca ctggaaatgg
     3181 caagcgctac cggagcactg cgcggaccac ctacaccttt gccaagccaa tggcggctaa
     3241 ctatctgaag aaccagccga tgtacgtgtt ccgtaagacg gagctcaagc actccaagac
     3301 cgagctcaac ttcaaggagt ggcaaaaggc ctttaccgat gtgatgggca tggacgagct
     3361 gtacaagggg tctggcggaa gcgggatggt gagcaagggc gaggaggata acatggcctc
     3421 tctcccagcg acacatgagt tacacatctt tggctccatc aacggtgtgg actttgacat
     3481 ggtgggtcag ggcaccggca atccaaatga tggttatgag gagttaaacc tgaagtccac
     3541 caagggtgac ctccagttct ccccctggat tctggtccct catatcgggt atggcttcca
     3601 tcagtacctg ccctaccctg acgggatgtc gcctttccag gccgccatgg tagatggctc
     3661 cggctaccaa gtccatcgca caatgcagtt tgaagatggt gcctccctta ctgttaacta
     3721 ccgctacacc tacgagggaa gccacatcaa aggagaggcc caggtgaagg ggactggttt
     3781 ccctgctgac ggtcctgtga tgaccaactc gctgaccgct gcggactggt gcaggtcgaa
     3841 gaagacttac cccaacgaca aaaccatcat cagtaccttt aagtggagtt acaccactgg
     3901 aaatggcaag cgctaccgga gcactgcgcg gaccacctac acctttgcca agccaatggc
     3961 ggctaactat ctgaagaacc agccgatgta cgtgttccgt aagacggagc tcaagcactc
     4021 caagaccgag ctcaacttca aggagtggca aaaggccttt accgatgtga tgggcatgga
     4081 cgagctgtac aagtcagcag gttccgcagg gatggtgagc aagggcgagg aggataacat
     4141 ggcctctctc ccagcgacac atgagttaca catctttggc tccatcaacg gtgtggactt
     4201 tgacatggtg ggtcagggca ccggcaatcc aaatgatggt tatgaggagt taaacctgaa
     4261 gtccaccaag ggtgacctcc agttctcccc ctggattctg gtccctcata tcgggtatgg
     4321 cttccatcag tacctgccct accctgacgg gatgtcgcct ttccaggccg ccatggtaga
     4381 tggctccggc taccaagtcc atcgcacaat gcagtttgaa gatggtgcct cccttactgt
     4441 taactaccgc tacacctacg agggaagcca catcaaagga gaggcccagg tgaaggggac
     4501 tggtttccct gctgacggtc ctgtgatgac caactcgctg accgctgcgg actggtgcag
     4561 gtcgaagaag acttacccca acgacaaaac catcatcagt acctttaagt ggagttacac
     4621 cactggaaat ggcaagcgct accggagcac tgcgcggacc acctacacct ttgccaagcc
     4681 aatggcggct aactatctga agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa
     4741 gcactccaag accgagctca acttcaagga gtggcaaaag gcctttaccg atgtgatggg
     4801 catggacgag ctgtacaagg gcagtggatc ctaagcgcca cttctaaata agcgaatttc
     4861 ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt atacaaattt
     4921 taaagtgact cttaggtttt aaaacgaaaa ttcttattct tgagtaactc tttcctgtag
     4981 gtcaggttgc tttctcaggt atagtatgag gtcgctctta ttgaccacac ctctaccggg
     5041 ctgcaggaat tcgatatcaa gcttatcgat accgtcgacc tcgagggggg gcccggtacc
     5101 agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg gtcatagctg
     5161 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5221 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5281 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5341 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5401 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5461 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5521 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5581 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5641 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5701 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5761 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5821 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5881 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5941 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6001 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6061 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6121 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6181 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6241 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6301 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6361 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6421 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6481 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6541 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6601 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6661 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6721 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6781 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6841 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6901 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6961 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7021 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7081 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7141 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7201 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7261 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     7321 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
