LOCUS       sequence-384006-        7469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Marker for yeast late Golgi/early endosome . Integration of 2xGFP 
            tag at SEC7 C terminus. Uses antibiotic resistance marker natMX6. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7469)
  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 7469)
  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: SEC7-2EGFP-ter-NAT
FEATURES             Location/Qualifiers
     source          1..7469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1103..1816
                     /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             1853..2569
                     /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             1853..2566
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2590..2777
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2645..2669)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2852..3971
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        2852..3195
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3196..3768
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      3774..3971
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    4034..4067
                     /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(5106..5125)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5107..5125)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5212..5229)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5383..5971)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5463..5482)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6142..6933,6934..7002))
                     /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     6765..6784
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7003..7107)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7175..7193
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7231..7253)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7353..7372
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7453..7469,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7453..7469,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttgatatt gcaaaacgta tctaaattca atgagtacca
       61 ttggaatcaa ataggtgacg tattcgataa attgtttgat ttaaccactg ctaatgaatt
      121 gttcgattat gatccgttac agcaaggaag aaaatcatct gtatctcatc accaaacaac
      181 taatgatacc agccaacact cagatgatga tagcaacgac agaagggaga acgattctaa
      241 tatcagtgag acggtcgaaa gagcgcatca agaagaatcc agcgaagacg ttggtggtga
      301 catggttgaa acactcaatg ggcaaactaa gcttaataat ggcaattccg ttccgacggt
      361 aaaggacgaa ttgaatccaa agcccgctag tctaagtatc cccaagaaaa ctaagcacat
      421 gaaacgaaac gaaagtaacg aagatatacg taggagaata aacatcaaga attctattgt
      481 tgtcaaatgt gttttacagc ttttgatgat agagctgctc aacgaattat tcgagaacga
      541 agattttgcc cactgtatcc cttacaagga agccattaga attacaagat tgttggagaa
      601 atcgtacgaa ttttctcgtg actttaatga agattatggg ttaagaacaa gactagtaga
      661 ggctcgtgta gttgataaaa taccaaacct gttgaaacaa gaaacgagtg ctgctgcagt
      721 tctccttgac attatgtttc agttgtatct gaacgatgat gagaagaagg ctgatttaat
      781 aactcgtctg atcaccattt gcatccaggt cgtagagggt tatgtttctt tggatgatag
      841 aaccatggaa cgtagtatca atgcctggcg ttcagtgata gttgaaatat tacaaggtta
      901 ctacgaattt gatgatgaag actttagact atactgtcct gctatgtatg ctctggtgat
      961 tcaaatatta gataagagcg ttccaacgga gttgagacat gctataaaac aatttctaag
     1021 cagagttggt gaattatacc tttctactga tgcttcgtac gctgcaggtc gacggatcgg
     1081 tgacggtgct ggtttaatta acatgtctaa aggtgaagaa ttattcactg gtgttgtccc
     1141 aattttggtt gaattagatg gtgatgttaa tggtcacaaa ttttctgtct ccggtgaagg
     1201 tgaaggtgat gctacttacg gtaaattgac cttaaaattt atttgtacta ctggtaaatt
     1261 gccagttcca tggccaacct tagtcactac tttaacttat ggtgttcaat gtttttctag
     1321 atacccagat catatgaaac aacatgactt tttcaagtct gccatgccag aaggttatgt
     1381 tcaagaaaga actatttttt tcaaagatga cggtaactac aagaccagag ctgaagtcaa
     1441 gtttgaaggt gataccttag ttaatagaat cgaattaaaa ggtattgatt ttaaagaaga
     1501 tggtaacatt ttaggtcaca aattggaata caactataac tctcacaatg tttacatcat
     1561 ggctgacaaa caaaagaatg gtatcaaagt taacttcaaa attagacaca acattgaaga
     1621 tggttctgtt caattagctg accattatca acaaaatact ccaattggtg atggtccagt
     1681 cttgttacca gacaaccatt acttatccac tcaatctgcc ttatccaaag atccaaacga
     1741 aaagagagac cacatggtct tgttagaatt tgttactgct gctggtatta cccatggtat
     1801 tgatgaattg tacaaaattc cagttccaac gcgtgatcta gagaagccat tcatgtctaa
     1861 aggtgaagaa ttattcactg gtgttgtccc aattttggtt gaattagatg gtgatgttaa
     1921 tggtcacaaa ttttctgtct ccggtgaagg tgaaggtgat gctacttacg gtaaattgac
     1981 cttaaaattt atttgtacta ctggtaaatt gccagttcca tggccaacct tagtcactac
     2041 tttaacttat ggtgttcaat gtttttctag atacccagat catatgaaac aacatgactt
     2101 tttcaagtct gccatgccag aaggttatgt tcaagaaaga actatttttt tcaaagatga
     2161 cggtaactac aagaccagag ctgaagtcaa gtttgaaggt gataccttag ttaatagaat
     2221 cgaattaaaa ggtattgatt ttaaagaaga tggtaacatt ttaggtcaca aattggaata
     2281 caactataac tctcacaatg tttacatcat ggctgacaaa caaaagaatg gtatcaaagt
     2341 taacttcaaa attagacaca acattgaaga tggttctgtt caattagctg accattatca
     2401 acaaaatact ccaattggtg atggtccagt cttgttacca gacaaccatt acttatccac
     2461 tcaatctgcc ttatccaaag atccaaacga aaagagagac cacatggtct tgttagaatt
     2521 tgttactgct gctggtatta cccatggtat ggatgaatta tataaataag gcgcgccact
     2581 tctaaataag cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa
     2641 ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg
     2701 agtaactctt tcctgtaggt caggttgctt tctcaggtat agtatgaggt cgctcttatt
     2761 gaccacacct ctaccggcag atccgctagg gataacaggg taatatagat ctgtttagct
     2821 tgcctcgtcc ccgccgggtc acccggccag cgacatggag gcccagaata ccctccttga
     2881 cagtcttgac gtgcgcagct caggggcatg atgtgactgt cgcccgtaca tttagcccat
     2941 acatccccat gtataatcat ttgcatccat acattttgat ggccgcacgg cgcgaagcaa
     3001 aaattacggc tcctcgctgc agacctgcga gcagggaaac gctcccctca cagacgcgtt
     3061 gaattgtccc cacgccgcgc ccctgtagag aaatataaaa ggttaggatt tgccactgag
     3121 gttcttcttt catatacttc cttttaaaat cttgctagga tacagttctc acatcacatc
     3181 cgaacataaa caaccatggg taccactctt gacgacacgg cttaccggta ccgcaccagt
     3241 gtcccggggg acgccgaggc catcgaggca ctggatgggt ccttcaccac cgacaccgtc
     3301 ttccgcgtca ccgccaccgg ggacggcttc accctgcggg aggtgccggt ggacccgccc
     3361 ctgaccaagg tgttccccga cgacgaatcg gacgacgaat cggacgacgg ggaggacggc
     3421 gacccggact cccggacgtt cgtcgcgtac ggggacgacg gcgacctggc gggcttcgtg
     3481 gtcgtctcgt actccggctg gaaccgccgg ctgaccgtcg aggacatcga ggtcgccccg
     3541 gagcaccggg ggcacggggt cgggcgcgcg ttgatggggc tcgcgacgga gttcgcccgc
     3601 gagcggggcg ccgggcacct ctggctggag gtcaccaacg tcaacgcacc ggcgatccac
     3661 gcgtaccggc ggatggggtt caccctctgc ggcctggaca ccgccctgta cgacggcacc
     3721 gcctcggacg gcgagcaggc gctctacatg agcatgccct gcccctaatc agtactgaca
     3781 ataaaaagat tcttgttttc aagaacttgt catttgtata gtttttttat attgtagttg
     3841 ttctatttta atcaaatgtt agcgtgattt atattttttt tcgcctcgac atcatctgcc
     3901 cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg
     3961 tcgctatact gctgtcgatt cgatactaac gccgccatcc agtgtcgaaa acgagctctc
     4021 gagaaccctt aatataactt cgtataatgt atgctatacg aagttattag gtgatatcag
     4081 atccactagt ggcctatgcg gccgcggcag attaaaatat gcttagttgt agaacacaga
     4141 aaaaatgaga aggttgatat acttttactc atattaatcc tgatttagtg gcggtgatcc
     4201 tttttagatt cgtttaattc ataaacaagg tacatcatat agcaataaca aatatgtaaa
     4261 ctatgtagaa agacttatca agtttagcca ttttggtcct cattcatggg cattttatac
     4321 taacttcatt tttgccgaaa taagaaagga tgtaatctta acctaaaata tgtaagcaat
     4381 taagggcgca cgtagtcact tgcgaacaaa taggaaaaga aagaaacaag cacggatact
     4441 gaatgactga gtgtattact acacgattta tctcaacgaa tttaagtgtg gaggacgatc
     4501 ggctgcacgt aggagtcaac gtttttttac tgtgttcatc acaaaaccat taagctgagt
     4561 catccatatt gcaaacaata aggtagcacc gcttccatca aaacactaga acataccgat
     4621 gttttcacgg atttattgga aacaaatagc agatgagggc gtttttactt gtaatgaagg
     4681 cgaatgagaa ggcaaggctt acaggcttgt tgtttatgcc aagcatcaat actcgctatg
     4741 gaggagcgcc atcagcgata acaacaaagt ttgcaagtgc ttgcttggat ataaaaggct
     4801 tgacgcttaa gataaatagg gccatacttg cttgctttta ctatttttgt tcctcataag
     4861 cctgattgac tacgctatta taaaccccta cattatccta taatcaactt cacttgtgta
     4921 acgttgaatg agttcaactg agtaatactc ggtcggaaat atacgctcca tctggcagga
     4981 aacataggct gcatttatat cttgcacaat tatgcccttc gtaggtgtat gttttcatgt
     5041 tttccattat gtcaatgtag atgtatcttt ggacaggcaa cttttggccg cggatctgcc
     5101 ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat
     5161 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5221 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5281 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5341 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5401 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5461 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5521 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5581 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5641 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5701 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5761 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5821 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5881 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5941 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6001 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6061 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6121 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6181 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6241 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6301 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6361 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6421 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6481 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6541 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6601 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6661 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6721 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6781 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6841 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6901 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6961 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7021 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7081 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     7141 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     7201 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     7261 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     7321 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     7381 gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat
     7441 catacacata cgatttaggt gacactata
//
