LOCUS       Exported                6682 bp ds-DNA     circular SYN 06-JAN-2025
DEFINITION  Plasmid for replacing Vrg4 with msGFP2-Vrg4 by gene replacement.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6682)
  AUTHORS   Glick Lab
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Oct 2, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Linearize with MluI for integration at VRG4.
FEATURES             Location/Qualifiers
     source          1..6682
                     /organism="synthetic DNA construct"
                     /lab_host="Saccharomyces cerevisiae"
                     /mol_type="other DNA"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    234..719
                     /label=5' VRG4
     CDS             720..1427
                     /codon_start=1
                     /product="yeast codon-optimized msGFP2"
                     /label=msGFP2
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITGGSGGS"
     CDS             1428..2438
                     /codon_start=1
                     /label=VRG4
                     /translation="SELKTGHAGHNPWASVANSGPISILSYCGSSILMTVTNKFVVNLK
                     DFNMNFVMLFVQSLVCTITLIILRILGYAKFRSLNKTDAKNWFPISFLLVLMIYTSSKA
                     LQYLAVPIYTIFKNLTIILIAYGEVLFFGGSVTSMELSSFLLMVLSSVVATWGDQQAVA
                     AKAASLAEGAAGAVASFNPGYFWMFTNCITSALFVLIMRKRIKLTNFKDFDTMFYNNVL
                     ALPILLLFSFCVEDWSSVNLTNNFSNDSLTAMIISGVASVGISYCSGWCVRVTSSTTYS
                     MVGALNKLPIALSGLIFFDAPRNFLSILSIFIGFLSGIIYAVAKQKKQQAQPLRK"
     misc_feature    2439..3169
                     /label=3' VRG4
     primer_bind     complement(3175..3191)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3659..4462)
                     /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(4463..4683)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        4776..4880
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4881..5741
                     /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"
     rep_origin      5912..6500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgt
      241 aatggaccat ggcaatgtgc gccatatata tatgcggctc ccttatgagt agaagaaaat
      301 atatgactgg tggtacaaag tggcatggtg gtatatagat gtaggaacac catttgagtt
      361 tgccataatg attaaatttc cctttttata aagaattagc cgcccataac acgaaaaata
      421 tcacagttga cgaaagaaga cacgtcgcct aaacgcgtaa tgaatcgcaa tatacgaaaa
      481 gctaaacggg tttttcagaa tcctcttacc cagccttgga taatagtttt ggccgaaaaa
      541 aaaaaaaaga aagaaaaaaa ttgaaatttc gcgatccgaa caaacaatga acagaaaaac
      601 agaatatata taacgctgcc aagcctattt cttgttgccg attaattgtt gacatcctct
      661 taattaccaa aagagcctaa gaaaacaaac acactaacca cacagtatct ttcgcccgaa
      721 tggatagcac tgagagcttg ttcacaggtg ttgttcctat cttggtcgaa ttagacggtg
      781 acgtaaacgg tcataagttc agtgttagag gtgaaggtga aggtgacgct actaacggta
      841 aattgacatt gaagtttatt tgtactacag gtaaattgcc agttccttgg ccaactttgg
      901 ttactacatt gacatacggt gttcaatgtt tttcaagata cccagatcat atgaagcaac
      961 atgatttctt taagtctgct atgccagaag gttacgttca agaaagaact atcactttta
     1021 aagatgatgg tacttacaag acaagagcag aggttaagtt cgaaggtgac actttggtta
     1081 acagaatcga attgaagggt atcgatttta aagaagatgg taatattttg ggtcataaat
     1141 tggaatacaa ctacaactca cataacgttt acattacagc tgataaacaa aagaatggta
     1201 ttaaagcaaa cttcaagatc agacataatg ttgaagatgg ttctgttcaa ttagcagatc
     1261 attaccaaca aaacactcca attggtgacg gtccagtttt gttaccagat aaccattact
     1321 tgtctacaca atcaaaattg tctaaagatc caaacgaaaa gagagatcat atggttttgt
     1381 tagaatttgt tactgctgct ggtatcacag gtggcagtgg cggttcttca gaattgaaaa
     1441 caggtcatgc aggccataac ccttgggctt cagttgccaa ttccggtccg atctctattt
     1501 tatcctactg tggttcctct attttaatga cggtgactaa caagttcgtc gtcaatttga
     1561 aggatttcaa catgaacttt gtcatgcttt tcgtgcaatc tttggtttgt actataacct
     1621 tgattatcct acgtatactg ggctatgcga agttccgttc attaaacaaa acagacgcca
     1681 agaactggtt ccctatttcc tttttactgg tcttgatgat ctacacctct tcgaaggctt
     1741 tacaatactt ggctgttcca atttacacca ttttcaagaa tttgactatt atcttgattg
     1801 cttatggtga ggttctcttt tttggtggct ctgtcacctc catggaattg tcatcatttt
     1861 tgttgatggt cctttcttct gtcgttgcaa cttggggtga ccagcaagct gtggctgcca
     1921 aggctgcttc attggctgaa ggagcagccg gtgctgttgc ctcctttaac ccaggttatt
     1981 tctggatgtt caccaactgt atcacttctg cattattcgt tcttataatg agaaagagaa
     2041 ttaagttaac taacttcaag gatttcgaca ctatgtttta caacaatgtt ttggctctac
     2101 ctattctatt gctgttttct ttctgtgtgg aagattggtc ttcagttaat ttgaccaata
     2161 acttttctaa cgattcgcta actgctatga tcatcagtgg tgttgcatcc gtcggtattt
     2221 cttactgttc cggttggtgt gttcgtgtta cttcgtctac tacatattcg atggtagggg
     2281 ctttgaacaa gctgccaatt gccttgtctg gtttgatttt ctttgatgct ccaagaaact
     2341 tcttatctat tctctccatt tttattggtt tcctatcagg tattatttat gctgttgcca
     2401 aacaaaagaa gcaacaagcc caacctttac gtaaatgaga acttacgggg ggtgcaattt
     2461 attttttttt tttggtttat tttattttat agaggcatct aatgcaagta gatttatata
     2521 caattatact taaaattgat ataccttaga acaggtcgga cacaacgtgt ggtaactcca
     2581 atatatcact aatgacaata gcgccttctg gtgtttgccc caaaatttca tttacttcat
     2641 tctcaactaa atgaatacat gttttcatgc ccaatttgat acctgtctcg atgtttttcc
     2701 cactgtcatc tataaaataa gcattctcgt atcttgccaa cccgctttcc ttcatggctt
     2761 tttcaaatgc tttaacatgt ggtttgcaga ccagcgtatc tgtcctagag tagtcacaat
     2821 atgtcaatcc atcaaataaa tccgctatac ccaacaatct caggcaacgt attgcgtggt
     2881 ttttataagc gttggtgaaa agccacaatt tatcaatctt tcccgattgt ctcaatctca
     2941 acagcatatt ccttaatggt atatcaggtt tcaagatatc ttgtagtggt aaggaatcat
     3001 ccacaagccg gttatattcc agtgcgttca ctttgtggaa cattacaagt ccccttatgg
     3061 caagtccata ctctttgtag taagagttat tcaaaacgtg agcatcttca ggtgatagtt
     3121 tcaagtgtgt ttgaaagaac ctcagtatag attgttgcat aagatcgtga attcactggc
     3181 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     3241 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     3301 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca
     3361 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     3421 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3481 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3541 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc gggtaataac tgatataatt
     3601 aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt
     3661 agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac
     3721 cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag
     3781 atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt
     3841 catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca
     3901 tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag
     3961 tacccttagt atattctcca gtagataggg agcccttgca tgacaattct gctaacatca
     4021 aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac
     4081 ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac
     4141 ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga
     4201 gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa
     4261 aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa
     4321 ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct
     4381 tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt
     4441 ccttatatgt agctttcgac atgatttatc ttcgtttcct gcatgttttt gttctgtgca
     4501 gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata
     4561 ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa
     4621 aaaaatttca aagaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaatt
     4681 gaaaagctgg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4741 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4801 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4861 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4921 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4981 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5041 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5101 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5161 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5221 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5281 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5341 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5401 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5461 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5521 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5581 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5641 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5701 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5761 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5821 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5881 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5941 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6001 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     6061 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6121 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6181 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6241 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6301 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6361 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6421 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6481 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6541 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6601 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6661 cagtgagcga ggaagcggaa ga
//
