LOCUS       sequence-384005-        7813 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Integration of 2xGFP tag at IRE1 C terminus. Potential indicator of 
            UPR activation. Uses antibiotic resistance marker kanMX4. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7813)
  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 7813)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: IRE1-2EGFP-ter-kanMX4
FEATURES             Location/Qualifiers
     source          1..7813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1121..1834
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1871..2584
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2614..2801
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2669..2693)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    2853..2886
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            2941..4297
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2941..3284
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3285..4094
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3352..3371)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      4100..4297
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    4360..4393
                     /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(5450..5469)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5451..5469)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5556..5573)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5727..6315)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5807..5826)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6486..7277,7278..7346))
                     /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     7109..7128
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7347..7451)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7519..7537
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7575..7597)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7697..7716
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7797..7813,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7797..7813,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttattaca gaaagagtat aatccaattt cgttattgag
       61 acaaatagcg tccggggtag cacatttaca ttctttaaag attatccatc gagatttaaa
      121 gcctcaaaat attctcgttt ctacttcgag taggtttact gccgatcagc aaacaggagc
      181 agaaaatctt cgaattttga tatcagactt tggtctttgc aaaaaactag actctggtca
      241 gtcttcattt agaacaaatt tgaataaccc ttctggcaca agtggttgga gggccccaga
      301 gctgcttgaa gaatcaaaca atttgcagtg ccaagtcgaa acggaacact cttctagtag
      361 gcatacagta gtttcatctg attcttttta tgatccgttc accaagagga ggctaacaag
      421 atctattgat attttttcta tgggatgtgt attctattat atcctatcca aagggaagca
      481 tccatttgga gataaatatt cacgtgaaag caatatcata agaggaatat tcagtcttga
      541 tgaaatgaaa tgtctacatg atagatcctt aattgcagaa gctacagatc tgatctccca
      601 aatgattgat cacgatccgt taaaaagacc tactgctatg aaagttctaa ggcatccgtt
      661 gttttggcca aagtcgaaaa aattggagtt ccttttaaaa gttagtgata ggcttgaaat
      721 tgaaaacaga gaccctccaa gtgccctgtt aatgaaattt gacgccggtt ctgactttgt
      781 aatacccagt ggagattgga ctgtcaagtt tgataaaaca ttcatggaca accttgaaag
      841 gtacagaaaa taccattcat caaagttaat ggatctatta agagcactta ggaataaata
      901 tcatcatttt atggatttac ctgaagatat agcagaacta atggggccgg tacccgatgg
      961 attttacgat tacttcacca agcgttttcc aaacctatta ataggtgttt atatgattgt
     1021 caaggaaaat ttaagtgacg atcaaatttt acgtgaattt ttgtattcag cttcgtacgc
     1081 tgcaggtcgt gggattcctg ggttaattaa catcgctagc atgagtaaag gagaagaact
     1141 tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt
     1201 ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat
     1261 ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt tcacttatgg
     1321 tgttcaatgc ttttcaagat acccagatca tatgaaacgg catgactttt tcaagagtgc
     1381 catgcccgaa ggttatgtac aggaaagaac tatatttttc aaagatgacg ggaactacaa
     1441 gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg
     1501 tattgatttt aaagaagatg gaaacattct tggacacaaa ttggaataca actataactc
     1561 acacaatgta tacatcatgg cagacaaaca aaagaatgga atcaaagtta acttcaaaat
     1621 tagacacaac attgaagatg gaagcgttca actagcagac cattatcaac aaaatactcc
     1681 aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtccacac aatctgccct
     1741 ttcgaaagat cccaacgaaa agagagacca catggtcctt cttgagtttg taacagctgc
     1801 tgggattaca catggcatgg atgaactata caaaattcca gttccaacgc gtgatctaga
     1861 gaagccattc atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga
     1921 attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc
     1981 aacatacgga aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg
     2041 gccaacactt gtcactactt tcacttatgg tgttcaatgc ttttcaagat acccagatca
     2101 tatgaaacgg catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac
     2161 tatatttttc aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga
     2221 tacccttgtt aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct
     2281 tggacacaaa ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca
     2341 aaagaatgga atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca
     2401 actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga
     2461 caaccattac ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca
     2521 catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata
     2581 caaacctagg tgaggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt
     2641 attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt
     2701 ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag
     2761 gtatagtatg aggtcgctct tattgaccac acctctaccg gcagatccgc tagggataac
     2821 agggtaatat ctgcaggtcg acaaccctta atataacttc gtataatgta tgctatacga
     2881 agttattagg tctagagatc tgtttagctt gcctcgtccc cgccgggtca cccggccagc
     2941 gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga
     3001 tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata
     3061 cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag
     3121 cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga
     3181 aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc
     3241 ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt aaggaaaaga
     3301 ctcacgtttc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     3361 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     3421 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     3481 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     3541 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggcaaa acagcattcc
     3601 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     3661 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     3721 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     3781 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataag ctcttgccat
     3841 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     3901 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     3961 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     4021 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     4081 atgagttttt ctaatcagta ctgacaataa aaagattctt gttttcaaga acttgtcatt
     4141 tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg tgatttatat
     4201 tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga aagtaatatc
     4261 atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat actaacgccg
     4321 ccatccagtg tcgaaaacga gctctcgaga acccttaata taacttcgta taatgtatgc
     4381 tatacgaagt tattaggtga tatcagatcc actagtggcc tatgcggccg cggtgttcat
     4441 gcccctctgc ttcttcttgg ttgattattg cattaatgtt actttgatca ttagtagtaa
     4501 ccatccataa tttttacgcc cttgcataaa tgaaaaaaag ttgtacttct tcatgaacct
     4561 atactacttt tctcatccct acataaacta cccgacagac atttaatttt ataaatacat
     4621 atctcataaa catcgaacat cgacatacta ctccagttat tcaacactga cttttttggt
     4681 ggaaagcttt cgttctgcaa ttcttatgca aataaaggta aacacacatg caaaagcgta
     4741 gacttcatca aaccaagaat ctatgacaat gtttgggacg gtaaactttc cacccaatag
     4801 ctcctttaat ctttgtgata cttcaaaagg gagatgggac ctaaccatta gcgttgtagc
     4861 caaaacgtag acgccagtca actctgacac caccagattt tttatcaaag atattccttt
     4921 ttgttcctga gaatagtttc tcttttttcc cttataggaa gagttattgg tgtactctgg
     4981 aagtacatta gcagtgtcat cattctgcat ggcgaaaagt gcaagaatct ggaactttat
     5041 gggcaaaagg gtcacaacgt aggagatggt ggtattgaca gcagaaagac agcaaagaaa
     5101 gagagaaatt gacaaaaata acgatatctg ttttgttaac gaatcgagat cgtgctggta
     5161 attgaacctg aaaaacgaaa aatccaaaat atttgctatc gtaatggcta aaggatcgga
     5221 tgctgcattt tcactaaaat tttcataatc gtagtcatct ggatatttga gcgagtggta
     5281 tatgatatag gggattcttt tcaggaaagt tattattagt ttatgaagac agtatatgat
     5341 aaacccgact tcaaaagctt tcttaaaagt gcgtacctga ggggactgtt gtaataatgc
     5401 ttccaatttc ccaagctcca gttgatacca gccgcggatc tgccggtctc cctatagtga
     5461 gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg
     5521 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5581 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5641 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5701 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5761 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5821 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5881 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5941 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6001 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6061 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6121 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6181 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6241 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6301 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6361 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6421 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6481 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6541 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6601 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6661 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6721 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6781 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6841 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6901 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6961 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7021 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7081 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7141 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7201 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7261 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7321 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7381 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7441 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     7501 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     7561 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     7621 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     7681 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatggacata
     7741 ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca catacgattt
     7801 aggtgacact ata
//
