LOCUS       sequence-382611-        7912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Marker for yeast endoplasmic reticulum (ER). Integration of 2xGFP 
            tag at EMC1 C terminus. Uses antibiotic resistance marker natMX6. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7912)
  AUTHORS   Li D, Yang SG, He CW, Zhang ZT, Liang Y, Li H, Zhu J, Su X, Gong Q, 
            Xie Z
  TITLE     Excess diacylglycerol at the endoplasmic reticulum disrupts 
            endomembrane homeostasis and autophagy.
  JOURNAL   BMC Biol. 2020 Aug 28;18(1):107. doi: 10.1186/s12915-020-00837-w.
  PUBMED    32859196
REFERENCE   2  (bases 1 to 7912)
  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: "2020-08-28"; volume: "18(1):107. doi"; pages: " 
            10.1186/s12915-020-00837-w"
COMMENT     Sequence Label: EMC1-2EGFP-ter-NAT
FEATURES             Location/Qualifiers
     source          1..7912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1156..1869
                     /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             1906..2622
                     /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             1906..2619
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2643..2830
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2698..2722)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2905..4024
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        2905..3248
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3249..3821
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      3827..4024
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    4087..4120
                     /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(5549..5568)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5550..5568)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5655..5672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5826..6414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5906..5925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6585..7376,7377..7445))
                     /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     7208..7227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7446..7550)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7618..7636
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7674..7696)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7796..7815
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7896..7912,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7896..7912,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcattta ctgttcaaat tgaatcccgg caagaatacg
       61 gatgtaccaa tagttgccaa caaccattct agttcccaca tattcgtcac agagcatgac
      121 acgaatggca tttatggcta cataatcgaa aacgatacgg taaaacaaac ttggaaaaaa
      181 gccgtaaatt cgaaagagaa aatggtggca tatagcaaga gggaaacaac aaacctaaac
      241 actcttggta ttacactagg tgacaaatcg gttctttata aatatttgta ccccaaccta
      301 gcggcttatc tgatcgctaa tgaagaacat catacaatca cttttaactt aattgatacc
      361 attacaggag aaatcctcat tacccaagag cacaaggatt ctccggattt taggtttcca
      421 atggatattg ttttcggtga atattgggtc gtttattcct atttcagttc tgaacctgtt
      481 ccagaacaaa agttagtagt ggtggaatta tatgagtcac taaccccaga tgagcgtttg
      541 tctaactcaa gcgacaattt ttcttatgat ccattgactg gacacattaa caaacctcaa
      601 tttcaaacta aacaattcat ttttcccgag attatcaaaa caatgtccat ttccaagaca
      661 acggatgata ttaccacaaa ggcaatcgtt atggaattag aaaatggaca aatcacctac
      721 ataccaaagc ttttattgaa tgcaagaggt aaaccagcag aagaaatggc caaggataag
      781 aaaaaagagt ttatggctac cccatacacg ccagttatcc caattaatga taatttcatt
      841 atcactcatt tcagaaatct attgccagga tccgattcgc agttgatctc catcccaacc
      901 aatctggaat ccacaagcat tatatgtgat ctaggccttg atgtattttg tacaaggatc
      961 acaccttcgg gccaatttga tttaatgagt cctactttcg aaaagggtaa attgcttatt
     1021 actatattcg tcttgttggt gatcacgtat tttatccgtc cttctgtttc aaacaagaag
     1081 ttgaaatccc aatggctaat taaagcttcg tacgctgcag gtcgacggat cggtgacggt
     1141 gctggtttaa ttaacatgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg
     1201 gttgaattag atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt
     1261 gatgctactt acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt
     1321 ccatggccaa ccttagtcac tactttaact tatggtgttc aatgtttttc tagataccca
     1381 gatcatatga aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa
     1441 agaactattt ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa
     1501 ggtgatacct tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac
     1561 attttaggtc acaaattgga atacaactat aactctcaca atgtttacat catggctgac
     1621 aaacaaaaga atggtatcaa agttaacttc aaaattagac acaacattga agatggttct
     1681 gttcaattag ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta
     1741 ccagacaacc attacttatc cactcaatct gccttatcca aagatccaaa cgaaaagaga
     1801 gaccacatgg tcttgttaga atttgttact gctgctggta ttacccatgg tattgatgaa
     1861 ttgtacaaaa ttccagttcc aacgcgtgat ctagagaagc cattcatgtc taaaggtgaa
     1921 gaattattca ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac
     1981 aaattttctg tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa
     2041 tttatttgta ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaact
     2101 tatggtgttc aatgtttttc tagataccca gatcatatga aacaacatga ctttttcaag
     2161 tctgccatgc cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac
     2221 tacaagacca gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta
     2281 aaaggtattg attttaaaga agatggtaac attttaggtc acaaattgga atacaactat
     2341 aactctcaca atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc
     2401 aaaattagac acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat
     2461 actccaattg gtgatggtcc agtcttgtta ccagacaacc attacttatc cactcaatct
     2521 gccttatcca aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact
     2581 gctgctggta ttacccatgg tatggatgaa ttatataaat aaggcgcgcc acttctaaat
     2641 aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa aaaataagtg
     2701 tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc ttgagtaact
     2761 ctttcctgta ggtcaggttg ctttctcagg tatagtatga ggtcgctctt attgaccaca
     2821 cctctaccgg cagatccgct agggataaca gggtaatata gatctgttta gcttgcctcg
     2881 tccccgccgg gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt
     2941 gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc
     3001 catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac
     3061 ggctcctcgc tgcagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt
     3121 ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc
     3181 tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat
     3241 aaacaaccat gggtaccact cttgacgaca cggcttaccg gtaccgcacc agtgtcccgg
     3301 gggacgccga ggccatcgag gcactggatg ggtccttcac caccgacacc gtcttccgcg
     3361 tcaccgccac cggggacggc ttcaccctgc gggaggtgcc ggtggacccg cccctgacca
     3421 aggtgttccc cgacgacgaa tcggacgacg aatcggacga cggggaggac ggcgacccgg
     3481 actcccggac gttcgtcgcg tacggggacg acggcgacct ggcgggcttc gtggtcgtct
     3541 cgtactccgg ctggaaccgc cggctgaccg tcgaggacat cgaggtcgcc ccggagcacc
     3601 gggggcacgg ggtcgggcgc gcgttgatgg ggctcgcgac ggagttcgcc cgcgagcggg
     3661 gcgccgggca cctctggctg gaggtcacca acgtcaacgc accggcgatc cacgcgtacc
     3721 ggcggatggg gttcaccctc tgcggcctgg acaccgccct gtacgacggc accgcctcgg
     3781 acggcgagca ggcgctctac atgagcatgc cctgccccta atcagtactg acaataaaaa
     3841 gattcttgtt ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt
     3901 ttaatcaaat gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc
     3961 gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat
     4021 actgctgtcg attcgatact aacgccgcca tccagtgtcg aaaacgagct ctcgagaacc
     4081 cttaatataa cttcgtataa tgtatgctat acgaagttat taggtgatat cagatccact
     4141 agtggcctat gcggccgcgg tagatattta catacacatt atatatatca gatacacatt
     4201 tcaaggctcc ttattttggc tttctttttt tttttttttt ttttccctgg aagacgtcat
     4261 actcgtggtt atataatctc ctacttcttt ggaaaatgga cacttaatca atgacggttg
     4321 atagcagtac gtgcactagc agactaatat gaagaagacg ttcgagcagt ttcgaaaaag
     4381 caatttacta tttcaggttc tcaaaggacc ccagcatcta gaatgtcaga agttatttgt
     4441 ccttgattct tcattcaatc caccacatct ggcccatttt caactactat cgcagactat
     4501 taaaaacttc aaattgaagg acacccgttc gcatgtttta ttactgttag cggtgaataa
     4561 tgcagataag ttgcctaagc cggcatcttt tccaactcgt ctggaaatga tgtgcttatt
     4621 cgctgactac cttcaggaga agctccccca atctgtagta tctgtcgggt tgactgtttt
     4681 ctcgaaattc atcgacaagg acaaaatatt acatgagcaa tttgttaaag gatgcagtgc
     4741 agatataggc tacttagttg gttttgatac aattgctagg atctttgatg aaaaatatta
     4801 tcatccttta aaaatcagtg atgtaatgga gagcttcatg tcgggatctc aattatattg
     4861 cttggcgaga ggcgattgcc atctcagtgc tgaatcgcaa ctaagatacg ccagtgacat
     4921 ccttgaggga aaattcgaac cggtaatacc aagagaatgg ggcgctagga ttcatgttat
     4981 gcaaaatgat tatccagcat taagaaatgt ttcatcatcc gagattagga acaaactgaa
     5041 gaatgggcaa gtggagagtt tgaaagacga gttgccattg tgcatatacg attatttgat
     5101 caataataag acaatatttg attgaaagag aaagatagaa attcaaatgc ctttcatgga
     5161 gatatatgta tatagataaa gataatatta ggaacaaaaa aaactttctg ttatactaga
     5221 ctagtaatct taaaacaata acatagcaac aaggcatgta ccaaaaagaa gggggtccaa
     5281 ttgcatgctt ggatttgctg agttcgcgta ttttgaaatc actcttttct tggagtcttc
     5341 accttcttgc gttaaatctt tgtacccttt ttctatgatt gtaccatcct gtttaacatt
     5401 cagcgtattt tccttcttac cagaaataca agtgaatttc ccacccctga tgattgatct
     5461 tccatttgat gggtttgtcc atttattgca atccaacacg tttgatgaac tcttaataaa
     5521 agcgctacct ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc
     5581 caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5641 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5701 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5761 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5821 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5881 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5941 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6001 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6061 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6121 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6181 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6241 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6301 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6361 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6421 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6481 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6541 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6601 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6661 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6721 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6781 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6841 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6901 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6961 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7021 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7081 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7141 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7201 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7261 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7321 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7381 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7441 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7501 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7561 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     7621 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     7681 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     7741 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     7801 tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac
     7861 aattaataca taaccttatg tatcatacac atacgattta ggtgacacta ta
//
