LOCUS       sequence-422256-        7368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of 2xGFP tag at YPQ2 C terminus, use auxotrophic marker 
            URA3(Kluyveromyces lactis) for selection. Marker for yeast vacuolar 
            membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7368)
  AUTHORS   He CW, Cui XF, Ma SJ, Xu Q, Ran YP, Chen WZ, Mu JX, Li H, Zhu J, 
            Gong Q, Xie Z
  TITLE     Membrane recruitment of Atg8 by Hfl1 facilitates turnover of 
            vacuolar membrane proteins in yeast cells approaching stationary 
            phase.
  JOURNAL   BMC Biol. 2021 Jun 4;19(1):117. doi: 10.1186/s12915-021-01048-7.
  PUBMED    34088313
REFERENCE   2  (bases 1 to 7368)
  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: "BMC Biol.";
            date: "2021-06-4"; volume: "19(1):117. doi"; pages: " 
            10.1186/s12915-021-01048-7"
COMMENT     Sequence Label: Ypq2-2GFP-URA
FEATURES             Location/Qualifiers
     source          1..7368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1281..1994
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGIDELYK"
     CDS             2031..2747
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2031..2744
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2768..2955
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2823..2847)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3238..3271
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      4226..4423
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    4477..4510
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5012..5031)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5013..5031)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5118..5135)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5289..5877)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5369..5388)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6048..6839,6840..6908))
                     /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     6671..6690
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6909..7013)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7081..7099
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7137..7159)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7259..7278
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7359..7368,1..9)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7359..7368,1..8)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 cactatagaa cgcggccgcc agctgaagct tggtaccagt gttatcacta agtatatctt
       61 ttagtatatc tcttcgcttg aggagggatc aaaaaaaaac aaacgtaaac gtgcacataa
      121 acgtcaagta ctttttgcag gcattaatat tcgtctccaa caaagacaaa taaagaagca
      181 atcttttcat taccttaagc cagtatctat tgatatcaat cgataaggtt cgttgttatc
      241 ggtgctaaaa attataaagt gaaaataaga atcttttcat gtcgtgctca aacggcatct
      301 ggcctactgt atctaatttg tgcggatcat tatccttctt cacctctgtt atttcattgt
      361 ttccacaaat aattgagact tatcgagata aatctgtcga tgggctatcg ccatattttt
      421 tgctggcttg gttatgtggg gatattacct cgctgatagg ggcaaagcta acagggcaat
      481 tgttatttca gatactttta gcaatttatt ttctattgaa tgattctttt gtatgtggtc
      541 aatattatta ctatggcgtg ctgcatgaaa ataaattggc gactgtcggc catgagccta
      601 aacctcttct tcccgaactg gttgaaaatg gtgagctttt gagggaggaa gaggacatga
      661 tacaaggtgg tagcagtgca gaaagcccta gaagcagtag aagaaggtct gccattacag
      721 cagccttggc tatagctcat actataagta cagctagcgc atatccgcta aatgtaggct
      781 ccacccaatc acaagtgggg cctcccggag atggtaaaaa cagccagctg ggtacaattt
      841 tatcttggat tggcgcttcc ttctacgtag gcgcacgtat cccacaacta atcaagaatt
      901 acaatagaaa gtctacagac ggcttatctc ccttcctatt tgctactaca ctactctgca
      961 atatcacgta taatttgagc atcttcacaa gctgtagatt tttagataat cagaacaaga
     1021 gggaattcat cgtgaatgag ttgcccttta tatttggaag tgctggaaca attgcgtttg
     1081 atttgatata cttctaccaa tactatatcc tttatgcaac tgatatgcaa ctacgtgaac
     1141 tggaaagaga actatacagt cctgaagaag acagtgcagc gcaactagtg acagaacgta
     1201 catcactgtt gtctggtgaa acgcaaacag cttcgtacgc tgcaggtcga cggatcggtg
     1261 acggtgctgg tttaattaac atgtctaaag gtgaagaatt attcactggt gttgtcccaa
     1321 ttttggttga attagatggt gatgttaatg gtcacaaatt ttctgtctcc ggtgaaggtg
     1381 aaggtgatgc tacttacggt aaattgacct taaaatttat ttgtactact ggtaaattgc
     1441 cagttccatg gccaacctta gtcactactt taacttatgg tgttcaatgt ttttctagat
     1501 acccagatca tatgaaacaa catgactttt tcaagtctgc catgccagaa ggttatgttc
     1561 aagaaagaac tatttttttc aaagatgacg gtaactacaa gaccagagct gaagtcaagt
     1621 ttgaaggtga taccttagtt aatagaatcg aattaaaagg tattgatttt aaagaagatg
     1681 gtaacatttt aggtcacaaa ttggaataca actataactc tcacaatgtt tacatcatgg
     1741 ctgacaaaca aaagaatggt atcaaagtta acttcaaaat tagacacaac attgaagatg
     1801 gttctgttca attagctgac cattatcaac aaaatactcc aattggtgat ggtccagtct
     1861 tgttaccaga caaccattac ttatccactc aatctgcctt atccaaagat ccaaacgaaa
     1921 agagagacca catggtcttg ttagaatttg ttactgctgc tggtattacc catggtattg
     1981 atgaattgta caaaattcca gttccaacgc gtgatctaga gaagccattc atgtctaaag
     2041 gtgaagaatt attcactggt gttgtcccaa ttttggttga attagatggt gatgttaatg
     2101 gtcacaaatt ttctgtctcc ggtgaaggtg aaggtgatgc tacttacggt aaattgacct
     2161 taaaatttat ttgtactact ggtaaattgc cagttccatg gccaacctta gtcactactt
     2221 taacttatgg tgttcaatgt ttttctagat acccagatca tatgaaacaa catgactttt
     2281 tcaagtctgc catgccagaa ggttatgttc aagaaagaac tatttttttc aaagatgacg
     2341 gtaactacaa gaccagagct gaagtcaagt ttgaaggtga taccttagtt aatagaatcg
     2401 aattaaaagg tattgatttt aaagaagatg gtaacatttt aggtcacaaa ttggaataca
     2461 actataactc tcacaatgtt tacatcatgg ctgacaaaca aaagaatggt atcaaagtta
     2521 acttcaaaat tagacacaac attgaagatg gttctgttca attagctgac cattatcaac
     2581 aaaatactcc aattggtgat ggtccagtct tgttaccaga caaccattac ttatccactc
     2641 aatctgcctt atccaaagat ccaaacgaaa agagagacca catggtcttg ttagaatttg
     2701 ttactgctgc tggtattacc catggtatgg atgaattata taaataaggc gcgccacttc
     2761 taaataagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat
     2821 aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag
     2881 taactctttc ctgtaggtca ggttgctttc tcaggtatag tatgaggtcg ctcttattga
     2941 ccacacctct accggcagat ccgctaggga taacagggta atatagatct gtttagcttg
     3001 cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc ctccttgaca
     3061 gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt tagcccatac
     3121 atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg cgaagcaaaa
     3181 attacggctc ctcgctgcgg acctgcgagc agggaaacgc tcccctcaca gacgcgtata
     3241 acttcgtata atgtatgcta tacgaagtta ttgaattgtc cccacgccgc gcccctgtag
     3301 agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact tccttttaaa
     3361 atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg acagtcaaca
     3421 ctaagaccta tagtgagaga gcagaaactc atgcctcacc agtagcacaa cgattatttc
     3481 gattaatgga actgaagaaa accaatttat gtgcatcaat tgatgttgat accactaagg
     3541 aattccttga attaattgat aaattgggtc cttatgtatg cttaatcaag actcatattg
     3601 atataatcaa tgatttttcc tatgaatcca ctattgaacc attattagaa ctttcacgta
     3661 aacatcaatt tatgattttt gaagatagaa aatttgctga tattggtaat accgtgaaga
     3721 aacaatatat tggtggagtt tataaaatta gtagttgggc agatattact aatgctcatg
     3781 gtgtcactgg gaatggagta gttgaaggat taaaacaggg agctaaagaa accaccacca
     3841 accaagagcc aagagggtta ttgatgttag ctgaattatc atcagtggga tcattagcat
     3901 atggagaata ttctcaaaaa actgttgaaa ttgctaaatc cgataaggaa tttgttattg
     3961 gatttattgc ccaacgtgat atgggtggac aagaagaagg atttgattgg cttattatga
     4021 cacctggagt tggattagat gataaaggtg atggattagg acaacaatat agaactgttg
     4081 atgaagttgt tagcactgga actgatatta tcattgttgg tagaggattg tttggtaaag
     4141 gaagagatcc agatattgaa ggtaaaaggt atagagatgc tggttggaat gcttatttga
     4201 aaaagactgg ccaattataa tcagtactga caataaaaag attcttgttt tcaagaactt
     4261 gtcatttgta tagttttttt atattgtagt tgttctattt taatcaaatg ttagcgtgat
     4321 ttatattttt tttcgcctcg acatcatctg cccagatgcg aagttaagtg cgcagaaagt
     4381 aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta
     4441 acgccgccat ccagtgtcga aaacgagctc gaattcataa cttcgtataa tgtatgctat
     4501 acgaagttat atcgatgata tcagatccac tagttagttt tccctacgga gtactaccat
     4561 ttatatttaa acaatgcact ctgctcttgg gatagtcctt gcgagaccct tttttcccaa
     4621 taggaatact acgttcgtac tctgtgtaaa agtacccaac tatagttagc gccagtttta
     4681 cgtaaaaccg tctatgtctt gtttgcattc gcaacagcta caaacacata tagtttgtca
     4741 cactaatatt aataatgaaa gttattatgt attcactgtg aacgcttcag tatacagtac
     4801 ggcagtaagt gtttcattct gttctggatt aataatcttt cactttgcct agccatttgg
     4861 gggcgggttt tggctgggtt agcttctcct tagtcatgga gaaaaaaaat cgcccgcaag
     4921 tatctcgaaa aaggaaaaaa tgcccacgcg tataagagtg gacccaactg ctcgagacta
     4981 gtggcctatg cggccgcgga tctgccggtc tccctatagt gagtcgtatt aatttcgata
     5041 agccaggtta acctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5101 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5161 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5221 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5281 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5341 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5401 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5461 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5521 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5581 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5641 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5701 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5761 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5821 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5881 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5941 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6001 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6061 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6121 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6181 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6241 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6301 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6361 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6421 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6481 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6541 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6601 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6661 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6721 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6781 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6841 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6901 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6961 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7021 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     7081 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     7141 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     7201 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     7261 gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc
     7321 tacaattaat acataacctt atgtatcata cacatacgat ttaggtga
//
