LOCUS       sequence-430388-        8594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Over-expression of GFP under TEF1 promoter, use auxotrophic marker 
            LEU2(Saccharomyces cerevisiae)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8594)
  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 8594)
  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: BS-ZJLEU-PTEF1-GFP
FEATURES             Location/Qualifiers
     source          1..8594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1061..1080)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1062..1080)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1087..1104)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1087..1103)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1096..1118)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1244..1699
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1331..1350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1541..1562
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2055..2074)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2174..2196
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2234..2252)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2337..2441
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2442..2510,2511..3302)
                     /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(2660..2679)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3473..4061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3962..3981
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4215..4232
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4349..4370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4385..4390,4391..4408,4409..4415)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4423..4439
                     /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     4428..4450
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4447..4463
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4447..4463
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4484..4502
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4790..5197
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     promoter        7017..7430
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             7473..8183
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(7500..7528)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     8123..8143
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      8212..8377
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(8267..8291)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 ttgtcaatac ttcatcatta atcaattcta tagttttgtt tttctcgttt atttccaaat
       61 ttaatgcatc aattttatta ttcaatttgt cgttgatttt ggttaatgat tttatggttt
      121 gatctctggc attgattgtt tgtgttagtt tttcattatt gataattaaa ttatttaagt
      181 tagttatcaa ctcggtgttt tcaagtttca agttttcaat ttctttagag tttattagat
      241 ttgtcaaagt ttctgaattg cttgattggt cctgtagaag agtatttgtt gttgtggata
      301 attgattcaa tttttgagac aattgctgga aggcgttgaa atatctagca tcaatctcat
      361 ggtttttttc ccgagagtct cgtagattca attgttttaa tatatcttgg gaccactctt
      421 gatttgaact catggaaatt aaactgggtg ttgtgttgtg gtgtaatgat tgtaccccct
      481 ttgcttataa ttgtgtggcc accgaagtcg gtgatgctgt cgccgaagaa gttaagaaaa
      541 tccttgctta aaaagattct ctttttttat gatatttgta cataaacttt ataaatgaaa
      601 ttcataatag aaacgacacg aaattacaaa atggaatatg ttcatagggt agacgaaact
      661 atatacgcaa tctacataca tttatcaaga aggagaaaaa ggaggatgta aaggaataca
      721 ggtaagcaaa ttgatactaa tggctcaacg tgataaggaa aaagaattgc actttaacat
      781 taatattgac aaggaggagg gcaccacaca aaaagttagg tgtaacagaa aatcatgaaa
      841 ctatgattcc taatttatat attggaggat tttctctaaa aaaaaaaaaa tacaacaaat
      901 aaaaaacact caatgacctg accatttgat ggagtttaag tcaatacctc cttgaaccat
      961 ttcccataat ggtgaaagtt ccctcaagaa ttttactctg tcagaaacgg ccttaacgac
     1021 gtaggccggc catttaaatg tttaaacggc cacgctggcc ccctatagtg agtcgtatta
     1081 caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     1141 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     1201 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc
     1261 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1321 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1381 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1441 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1501 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1561 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1621 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     1681 atattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     1741 cacaccgcat agggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt
     1801 attaggatct ctacccttgg cgaaaagtcc tctgccaaca atgatgatat ctgatccacc
     1861 acttacaact tcgtcgacgg ttctgtactg ctgacccaat atgcgtatat ataccaatct
     1921 aagtctgtgc tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt
     1981 tcaaggaaac cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga aaaggtggta
     2041 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     2101 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     2161 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     2221 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     2281 gtttcttaga cgtcggcgcg ccgaggacgt caggtggcac ttttcgggga aatgtgcgcg
     2341 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2401 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     2461 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     2521 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     2581 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     2641 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     2701 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2761 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2821 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2881 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2941 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     3001 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     3061 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     3121 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     3181 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     3241 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3301 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     3361 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3421 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3481 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3541 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3601 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3661 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3721 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3781 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3841 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3901 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3961 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4021 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4081 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4141 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4201 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     4261 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     4321 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     4381 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     4441 ttcacacagg aaacagctat gaccatgatt acgccaagct cggaattaac cctcactaaa
     4501 gggaacaaaa gctgggccgg ccatttaaat gtttaaacgg ccacgctggc cgaggagaac
     4561 ttctagtata tctacatacc taatattatt gccttattaa aaatggaatc ccaacgatta
     4621 catcaaaatc cacattctct tcaaaatcaa ttgtcctgta cttccttgtt catgtgtgtt
     4681 caaaaacgtt atatttatag gataattata ctctatttct caacaagtaa ttggttgttt
     4741 ggccgagcgg tctaaggcgc ctgattcaag aaatatcttg accgcagtta actgtgggaa
     4801 tactcaggta tcgtaagatg caagagttcg aatctcttag caaccattat ttttttcctc
     4861 aacataacga gaacacacag gggcgctatc gcacagaatc aaattcgatg actggaaatt
     4921 ttttgttaat ttcagaggtc gcctgacgca tatacctttt tcaactgaaa aattgggaga
     4981 aaaaggaaag gtgagagcgc cggaaccggc ttttcatata gaatagagaa gcgttcatga
     5041 ctaaatgctt gcatcacaat acttgaagtt gacaatatta tttaaggacc tattgttttt
     5101 tccaataggt ggttagcaat cgtcttactt tctaactttt cttacctttt acatttcagc
     5161 aatatatata tatatatttc aaggatatac cattctaatg tctgccccta agttaccaat
     5221 agatagcggt ttaatataag gtttcaacaa gaaatatttt gtgaaaatca aaagttaata
     5281 gtttcactgt cgaaaatatg aaagtttaag ttttcagttc atcccattgt agttatcaaa
     5341 tttctgtgtt tcccgtatat gtgtgctctg tattcatcgc tatcttgaaa ctggttatga
     5401 tcgttgcgca tgtgacctgt atcgctcaaa agagcccggc accggtaccg ggcaaaagcc
     5461 cctagcaagc taaaatgatt caactttcgt agtaatttgt tcaaccggtc acagtaaagt
     5521 caatgactaa cttttgaatc gaggatgtat ttccaattga aagcaatgta tatataaacc
     5581 attgtgttga agcttgtctt acagttttct tgtatcctcc cattgttcat atagtactta
     5641 aagtaccata atccttaaag aacaaacttg aaagagaata cactatacac aactaataaa
     5701 taaagaataa tcatggctgt gaccaagaca attgtagttc taccaggtga ccatgttggt
     5761 caagaaatca ctgaagaggc cattaaagtt ttaaatgcta ttcaggaatg tcgtccagac
     5821 aaggtcaatt tcaagtttga tcatcatttg atcggtggtg ctgcaattga tgccactggt
     5881 gttccattac cagacgaagc tttggaggcc tctaagaaag ctgatgccgt gcttctgggt
     5941 gctgttggtg gtccaaaatg gggtactggc gctgtcagac cagaacaagg tcttttgaaa
     6001 atccgtaagg agttgcaatt gtatgctaat ctaagaccat gtaattttgc atctgattcc
     6061 ttactagatc tatcgccatt gaagcctgaa attgcaagag gtacagattt cgtcgttgtt
     6121 agagaactag tgggtggtat ttatttcggg gagagaaaag aagatgaagg tgatggtgtc
     6181 gcctgggata gcgaaaagta ttctgtgccc gaagttcaaa gaatcactag aatggctgct
     6241 ttcatggctc tgcaacacaa cccaccttta cctatttggt ctctagataa agccaacgtt
     6301 ctagcatcat caagattatg gagaaaaaca gtggaagaga cgattaagaa tgaattccca
     6361 caattgactg ttcaacatca attgattgat tccgctgcta tgatcttggt taagaaccca
     6421 actcatctaa atggtattat tatcacaaac aatatgtttg gtgatattat ctccgatgaa
     6481 gcctccgtga tcccaggttc gttaggatta cttccatctg cctccttggc atctcttcca
     6541 gataagaata cggcatttgg tttatatgag ccatgtcatg gctctgctcc agacttacca
     6601 aagggaaagg ttaaccccgt agcaacaatc ctatctgctg ctatgatgtt gaagttgtct
     6661 ctggacctgt tcgaagaagg tgaaattata gaacaagcag tcaagaaagt gttggattct
     6721 ggtatcagaa cagcggatct aaagggtacc aactctacta ctgaggtcgg tgatgcagta
     6781 gcgaaagctg tcagggaact attagcttag acttagtaga tgtacagttg catacataag
     6841 tctgaacaga aagaagtaaa tatcatatct agttcggttt agaaatcagt gactatattg
     6901 gttttgacca gatcgcaatc cttgcaagca tttagtaatg actatcaaac cggtgaatca
     6961 tctgataagg ctatattagc ttgtgcattc gcatgtatcg ggaaacgaac ggtaccgggc
     7021 ccccacacac catagcttca aaatgtttct actccttttt tactcttcca gattttctcg
     7081 gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta aatttcccct
     7141 ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa gaaaaaagag
     7201 accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt
     7261 ttcttgaaaa tttttttttt tgattttttt ctctttcgat gacctcccat tgatatttaa
     7321 gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac
     7381 tttttttact tcttgctcat tagaaagaaa gcatagcaat ctaatctaag ttttaattac
     7441 aaatgctcga gcggatcccc gggttaatta acagtaaagg agaagaactt ttcactggag
     7501 ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg
     7561 gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg
     7621 gaaaactacc tgttccatgg ccaacacttg tcactacttt cacttatggt gttcaatgct
     7681 tttcaagata cccagatcat atgaaacggc atgacttttt caagagtgcc atgcccgaag
     7741 gttatgtaca ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg
     7801 aagtcaagtt tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta
     7861 aagaagatgg aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat
     7921 acatcatggc agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt agacacaaca
     7981 ttgaagatgg aagcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg
     8041 gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc
     8101 ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac
     8161 atggcatgga tgaactatac aaatagggat cctgagtaaa taagctgccg cgcgaatttc
     8221 ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt atacaaattt
     8281 taaagtgact cttaggtttt aaaacgaaaa ttcttattct tgagtaactc tttcctgtag
     8341 gtcaggttgc tttctcaggt atagcatgag gtcgctctta ttgaccacac cgagctctaa
     8401 gcaaatagct aaattatata cgaattaata ttatgattaa gtgtttacgt gagtgcgata
     8461 tttttattac tatcttatac agttgtatat actctataaa atgagttgtc tattaattaa
     8521 cgcgatgaat gctttctggg tttacctctc caacaactct agtttacttc tcaatacatt
     8581 caattgtatt tgat
//
