LOCUS       sequence-454833-        7466 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 7466)
  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 7466)
  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::URA3
FEATURES             Location/Qualifiers
     source          1..7466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             96..899
                     /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(1030..1485)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1167..1188)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1379..1398
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1611..1633
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1625..1642
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1626..1642
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1649..1668
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1649..1667
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1700..1716
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1700..1716
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             2365..2415
                     /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             2440..3147
                     /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      2695..2704
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3166..3873
                     /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      3421..3430
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3892..4599
                     /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      4147..4156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     terminator      4632..4819
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4687..4711)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(4847..4863)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4848..4864)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(4892..4910)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4931..4947)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4931..4947)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4944..4966)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4955..4971
                     /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(4979..4985,4986..5003,5004..5009))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5024..5045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5162..5179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5333..5921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5413..5432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6092..6883,6884..6952))
                     /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     6715..6734
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6953..7057)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7125..7143
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7181..7203)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7303..7322
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7346..7466,1..95)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
ORIGIN
        1 atatacgcat atgtagtgtt gaagaaacat gaaattgccc agtattctta acccaactgc
       61 acagaacaaa aacctgcagg aaacgaagat aaatcatgtc gaaagctaca tataaggaac
      121 gtgctgctac tcatcctagt cctgttgctg ccaagctatt taatatcatg cacgaaaagc
      181 aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa ggaattactg gagttagttg
      241 aagcattagg tcccaaaatt tgtttactaa aaacacatgt ggatatcttg actgattttt
      301 ccatggaggg cacagttaag ccgctaaagg cattatccgc caagtacaat tttttactct
      361 tcgaagacag aaaatttgct gacattggta atacagtcaa attgcagtac tctgcgggtg
      421 tatacagaat agcagaatgg gcagacatta cgaatgcaca cggtgtggtg ggcccaggta
      481 ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa ggaacctaga ggccttttga
      541 tgttagcaga attgtcatgc aagggctccc tatctactgg agaatatact aagggtactg
      601 ttgacattgc gaagagcgac aaagattttg ttatcggctt tattgctcaa agagacatgg
      661 gtggaagaga tgaaggttac gattggttga ttatgacacc cggtgtgggt ttagatgaca
      721 agggagacgc attgggtcaa cagtatagaa ccgtggatga tgtggtctct acaggatctg
      781 acattattat tgttggaaga ggactatttg caaagggaag ggatgctaag gtagagggtg
      841 aacgttacag aaaagcaggc tgggaagcat atttgagaag atgcggccag caaaactaaa
      901 aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat
      961 tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     1021 cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     1081 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     1141 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     1201 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     1261 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     1321 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     1381 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     1441 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
     1501 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     1561 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     1621 acgttgtaaa acgacggcca gtgaattgta atacgactca ctatagggcg aattggagct
     1681 ccaccgcggt ggcggccgct ctagaactag tggatcccgg gtttgcgttt aagatgccaa
     1741 gattcatcag ctgtcatgcc aaggccattg attacccaca atcaaacata acctacatag
     1801 gaattgaccc taaaccagca aactacaacc aaacacaact ttcaaaatac tatgatgatc
     1861 tagtacaaat ggaagataaa aatgcactaa gcttattttc ttcagattgg tacgctacaa
     1921 aggatagatt actcaccaag aaaagatctc gcaacccatt taacagaact gcaccgtacg
     1981 ctcagaatat tttctgcaaa gaaaacggca agaggatcga gggaatcgag gatgacgagg
     2041 aatattttga aacaaaaatt aagtgtaaaa tgccatggtc gtcgccaaga caataaacgt
     2101 gtgggagtat tgcaaaaaca tagtatataa gcgtaaatca ctatataaag cccaaagaag
     2161 ttagcactaa agcatcattc atcgttcaaa actcctctct caagtgagag aattcggcgc
     2221 tgaaaaaaag ctttgtcaaa accaagaaaa aaacgtagga ttggacacga ggttcttttt
     2281 ttaatgtaaa caagaagtgg cactatagct gtgaaacttc caatacaatt gtataccaaa
     2341 taagctatta caaattgatt taaaatgggt gtcgcagatt tgatcaagaa attcgaaagc
     2401 atctcaaagg aagaaggtga cggtgctggt ttaattaaca tggtgagcaa gggcgaggag
     2461 gataacatgg cctctctccc agcgacacat gagttacaca tctttggctc catcaacggt
     2521 gtggactttg acatggtggg tcagggcacc ggcaatccaa atgatggtta tgaggagtta
     2581 aacctgaagt ccaccaaggg tgacctccag ttctccccct ggattctggt ccctcatatc
     2641 gggtatggct tccatcagta cctgccctac cctgacggga tgtcgccttt ccaggccgcc
     2701 atggtagatg gctccggcta ccaagtccat cgcacaatgc agtttgaaga tggtgcctcc
     2761 cttactgtta actaccgcta cacctacgag ggaagccaca tcaaaggaga ggcccaggtg
     2821 aaggggactg gtttccctgc tgacggtcct gtgatgacca actcgctgac cgctgcggac
     2881 tggtgcaggt cgaagaagac ttaccccaac gacaaaacca tcatcagtac ctttaagtgg
     2941 agttacacca ctggaaatgg caagcgctac cggagcactg cgcggaccac ctacaccttt
     3001 gccaagccaa tggcggctaa ctatctgaag aaccagccga tgtacgtgtt ccgtaagacg
     3061 gagctcaagc actccaagac cgagctcaac ttcaaggagt ggcaaaaggc ctttaccgat
     3121 gtgatgggca tggacgagct gtacaagggg tctggcggaa gcgggatggt gagcaagggc
     3181 gaggaggata acatggcctc tctcccagcg acacatgagt tacacatctt tggctccatc
     3241 aacggtgtgg actttgacat ggtgggtcag ggcaccggca atccaaatga tggttatgag
     3301 gagttaaacc tgaagtccac caagggtgac ctccagttct ccccctggat tctggtccct
     3361 catatcgggt atggcttcca tcagtacctg ccctaccctg acgggatgtc gcctttccag
     3421 gccgccatgg tagatggctc cggctaccaa gtccatcgca caatgcagtt tgaagatggt
     3481 gcctccctta ctgttaacta ccgctacacc tacgagggaa gccacatcaa aggagaggcc
     3541 caggtgaagg ggactggttt ccctgctgac ggtcctgtga tgaccaactc gctgaccgct
     3601 gcggactggt gcaggtcgaa gaagacttac cccaacgaca aaaccatcat cagtaccttt
     3661 aagtggagtt acaccactgg aaatggcaag cgctaccgga gcactgcgcg gaccacctac
     3721 acctttgcca agccaatggc ggctaactat ctgaagaacc agccgatgta cgtgttccgt
     3781 aagacggagc tcaagcactc caagaccgag ctcaacttca aggagtggca aaaggccttt
     3841 accgatgtga tgggcatgga cgagctgtac aagtcagcag gttccgcagg gatggtgagc
     3901 aagggcgagg aggataacat ggcctctctc ccagcgacac atgagttaca catctttggc
     3961 tccatcaacg gtgtggactt tgacatggtg ggtcagggca ccggcaatcc aaatgatggt
     4021 tatgaggagt taaacctgaa gtccaccaag ggtgacctcc agttctcccc ctggattctg
     4081 gtccctcata tcgggtatgg cttccatcag tacctgccct accctgacgg gatgtcgcct
     4141 ttccaggccg ccatggtaga tggctccggc taccaagtcc atcgcacaat gcagtttgaa
     4201 gatggtgcct cccttactgt taactaccgc tacacctacg agggaagcca catcaaagga
     4261 gaggcccagg tgaaggggac tggtttccct gctgacggtc ctgtgatgac caactcgctg
     4321 accgctgcgg actggtgcag gtcgaagaag acttacccca acgacaaaac catcatcagt
     4381 acctttaagt ggagttacac cactggaaat ggcaagcgct accggagcac tgcgcggacc
     4441 acctacacct ttgccaagcc aatggcggct aactatctga agaaccagcc gatgtacgtg
     4501 ttccgtaaga cggagctcaa gcactccaag accgagctca acttcaagga gtggcaaaag
     4561 gcctttaccg atgtgatggg catggacgag ctgtacaagg gcagtggatc ctaagcgcca
     4621 cttctaaata agcgaatttc ttatgattta tgatttttat tattaaataa gttataaaaa
     4681 aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa ttcttattct
     4741 tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagtatgag gtcgctctta
     4801 ttgaccacac ctctaccggg ctgcaggaat tcgatatcaa gcttatcgat accgtcgacc
     4861 tcgagggggg gcccggtacc agcttttgtt ccctttagtg agggttaatt tcgagcttgg
     4921 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4981 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5041 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5101 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5161 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5221 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5281 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5341 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5401 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5461 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5521 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5581 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5641 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5701 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5761 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5821 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5881 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5941 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6001 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6061 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6121 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6181 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6241 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6301 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6361 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6421 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6481 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6541 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6601 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6661 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6721 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6781 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6841 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6901 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6961 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7021 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7081 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     7141 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     7201 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     7261 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     7321 tactgagagt gcaccatacc accttttcaa ttcatcattt tttttttatt cttttttttg
     7381 atttcggttt ccttgaaatt tttttgattc ggtaatctcc gaacagaagg aagaacgaag
     7441 gaaggagcac agacttagat tggtat
//
