LOCUS       sequence-382499-        8300 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Integration of 2xGFP tag at PKC1 C terminus. Uses auxotrophic marker
            TRP1(Kluyveromyces lactis). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8300)
  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 8300)
  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: PKC1-2GFP-ter-TRP1
FEATURES             Location/Qualifiers
     source          1..8300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2329..3042
                     /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             3079..3792
                     /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"
     protein_bind    3812..3845
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4848..4881
                     /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(5937..5956)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5938..5956)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6043..6060)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6214..6802)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6294..6313)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6973..7764,7765..7833))
                     /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     7596..7615
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7834..7938)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8006..8024
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8062..8084)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8184..8203
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(8284..8300,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(8284..8300,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttgctatt atcaatagga aagaagagat attcgaacag
       61 catggccacc actttgtaca aaaatcattt tacaacatta tgtgctgtgc atattgtggt
      121 gatttcctcc ggtatactgg attccaatgt caagattgta aatttttatg tcacaaaaaa
      181 tgttacacca atgttgttac taagtgtatt gctaaaactt ctactgatac ggatccggac
      241 gaggcaaagt tgaaccaccg tattcctcat agattcctgc ctacttcaaa ccgtggtact
      301 aaatggtgct gtcactgtgg ttatatttta ccatggggta gacataaagt acgcaaatgt
      361 tctgaatgtg gtataatgtg tcatgctcaa tgtgctcatt tggttcccga tttctgtggc
      421 atgtcgatgg aaatggctaa taaaattctg aaaacgattc aagacacaaa acgtaatcaa
      481 gagaaaaaga agaggacagt tccgtcggca cagttggggt catctattgg tactgccaat
      541 ggtagtgatc tttccccatc caaattagct gaaagggcaa acgctccttt gcctcctcag
      601 ccaagaaaac atgataaaac tccttcgcct cagaaggtag gaagagattc tccaactaag
      661 cagcatgatc ctattattga taaaagaatt tcactacaaa cgcacgggcg tgagaaacta
      721 aataaattta tcgatgaaaa tgaggcctat ttaaatttta cagaaggtgc acaacaaacc
      781 gccgaatttt catcaccaga gaaaacactg gatccaacgt cgaatagacg ttccttggga
      841 ctcactgatt tatctattga gcatagtcaa acttgggaat ctaaagatga tttaatgaga
      901 gatgaacttg aactgtggaa agcacaacgt gaggaaatgg aacttgaaat aaaacaagat
      961 agtggggaaa tccaagagga tctagaagtt gatcacattg atctagagac aaaacagaaa
     1021 ctagactggg agaacaaaaa tgatttccgt gaagctgatt tgacaataga ttccacacac
     1081 acaaatccat tccgcgatat gaatagtgaa acctttcaaa ttgaacaaga ccatgcttca
     1141 aaggaagtac tacaagaaac agtgtccttg gctcctacaa gtactcatgc ctccagaacc
     1201 actgatcaac aatctccgca gaaatcacaa accagcacaa gcgcaaagca taaaaagaga
     1261 gcggctaaac gtcgtaaggt ttcattagat aatttcgtat tactcaaagt tcttggtaaa
     1321 ggtaattttg gtaaagttat tttatctaaa tcaaagaata ctgacaggct ttgtgccata
     1381 aaagttctga agaaagataa tattattcaa aatcatgacc tcgagagtgc aagagcagaa
     1441 aagaaagtat ttttgttagc cacaaaaact aaacacccat tcttaaccaa tctatactgc
     1501 tcttttcaaa ctgaaaaccg tatatatttt gctatggagt ttattggggg cggtgactta
     1561 atgtggcatg ttcaaaacca aagactatct gtaagaaggg ccaaatttta tgccgccgag
     1621 gtcttactgg ctctgaaata ttttcatgat aatggtgtaa tataccgtga tttgaagttg
     1681 gaaaacattc tattaactcc agaaggtcat ataaaaattg ccgattatgg tttgtgtaaa
     1741 gatgaaatgt ggtatggtaa tagaacttct acattttgtg gcacaccaga atttatggct
     1801 ccagaaattt taaaagagca agaatatacc aaagctgtcg attggtgggc atttggggtg
     1861 ctgctatatc aaatgctact gtgccaatct ccattctcag gagatgacga agatgaagtt
     1921 tttaacgcta tccttaccga tgaaccctta tacccaatag atatggcagg tgagattgta
     1981 caaatattcc aaggcctatt aacaaaagat cctgaaaaga ggttgggtgc tggtcccagg
     2041 gacgcagacg aagttatgga agaacctttc ttccgtaaca tcaactttga cgatatctta
     2101 aaccttcgcg tgaaaccacc ctacatccca gaaattaaat ctccggaaga tacatcatat
     2161 ttcgagcaag aattcacctc tgcgccaccc acacttacac ctctgccctc tgttttgaca
     2221 acgagccagc aagaagagtt tagaggattt tcctttatgc cagatgattt ggatttagct
     2281 tcgtacgctg caggtcgtgg gattcctggg ttaattaaca tcgctagcat gagtaaagga
     2341 gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg
     2401 cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt
     2461 aaatttattt gcactactgg aaaactacct gttccatggc caacacttgt cactactttc
     2521 acttatggtg ttcaatgctt ttcaagatac ccagatcata tgaaacggca tgactttttc
     2581 aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg
     2641 aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag
     2701 ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac
     2761 tataactcac acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac
     2821 ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa
     2881 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa
     2941 tctgcccttt cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta
     3001 acagctgctg ggattacaca tggcatggat gaactataca aaattccagt tccaacgcgt
     3061 gatctagaga agccattcat gagtaaagga gaagaacttt tcactggagt tgtcccaatt
     3121 cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa
     3181 ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct
     3241 gttccatggc caacacttgt cactactttc acttatggtg ttcaatgctt ttcaagatac
     3301 ccagatcata tgaaacggca tgactttttc aagagtgcca tgcccgaagg ttatgtacag
     3361 gaaagaacta tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt
     3421 gaaggtgata cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga
     3481 aacattcttg gacacaaatt ggaatacaac tataactcac acaatgtata catcatggca
     3541 gacaaacaaa agaatggaat caaagttaac ttcaaaatta gacacaacat tgaagatgga
     3601 agcgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt
     3661 ttaccagaca accattacct gtccacacaa tctgcccttt cgaaagatcc caacgaaaag
     3721 agagaccaca tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat
     3781 gaactataca aacctaggtg aggcgcgcca tataacttcg tataatgtat gctatacgaa
     3841 gttattaggt ctagagcggc ctcggaacga tcattcacta tatatatatc aatttatata
     3901 tacgtatgtg taattgaaga aagatacgtt ttttcctcta ttgagaggcc tgctggatga
     3961 atagctttac cttttctaaa tccttgatac catcagtctc tactcctcca cttacatcga
     4021 caccgatcgc atttggtaac atattaatgg caacagaaac gttatcagga ttcaatccac
     4081 cagcgataat gaattttatc tcgggatgac ttgcagacca actggaaatt gcactccaat
     4141 tcaatttctc accagtgcca ccttcaccag aatcgaacaa cgtcagcaca ttgtctacgt
     4201 gttcatacag gtccagtagt aattcacaat cctgtgggaa ctggaacctc ttaatgattg
     4261 gaattgaaga tgggatcaaa gatctgtatt ctttaatatc ttcatctcca tgtaattgta
     4321 tcacatctag attatattcg tggtacagtt gaaggacatc atcaacggac tgatttctaa
     4381 acaccccgac tagtttagta cctttcacgt tctcttgttg gtgaactgca gttgaaatac
     4441 ctttcgcaac agatgagtca atggttcttt tcctaccggg aacacaaatg atacctaagt
     4501 aatcagcacc atcatccaca gcagtctttg cagcttcaac ggtttgcaaa ccacacactt
     4561 taacgagcat cacagtagat ttcgcaagag ggtagctcgc tcagagtacc caagtaaatg
     4621 attagtaaac tgatgtttga tagttcaatt tttcaatgaa ataaccttat attaaaattg
     4681 atattactat tatacaaaaa taaagaataa aggatttgag tttatacata aaataccatt
     4741 attatttgtt cagtgagaga tactggggta tatgggatgt gtgtagtgat accatgcaat
     4801 catgtatcaa acatgggccc ggtaccgagc tctcgagaac ccttaatata acttcgtata
     4861 atgtatgcta tacgaagtta ttaggtgata tcagatccac tagtggccta tgcggccgcg
     4921 gtgaagaaaa ggtcatgcca tgatttattt aattttatat aaaacatcta agcggaaaag
     4981 ttgaactgtt ccgcttcata ggaggagagg gtttttttaa tgagattttg ttacataaac
     5041 tgaaactatc cattttgatg caatgcctct tttttgttcg tacctatctt ggagcatgca
     5101 tatgaaaaac ctcgtttaag cgatgcgatg cgaagccgta tgttcaacaa tgcgcattct
     5161 gtttacatta ttaaggaagc atttaagaaa cagctcagta ttatattact actaacatat
     5221 ttctaaagtt gacagattat aaaagttaca aaattgtcaa ttgaacagat acaaaaattt
     5281 tataaatgtt taagaaaagc aggcatttga agaatgtgtc aaatgctatc aaatccgcga
     5341 gagttcatga cgtctcaaat ggtattaact ccaaattttt cgatactaag aaaatatgca
     5401 catacggcat aaatggacga atcactgtta ccacattcga ctatactcaa agccttttgg
     5461 cagttgcaac tactgctggg gagatacatg tttatgggca aaagcaaata gaggtcgtat
     5521 ttacgttaaa aaatcgacct caaattaaac acatgcggtt tattaaagga atttatctga
     5581 tagctgtaga tgagaagagc aacataatag ttctttcagt acactcgaaa cagattctaa
     5641 ctactgtttt ctgtccaaac agcatcactt gtattgagac tgatccgtcc ttggattgga
     5701 tgctgattgg cctcgagagt ggatccatat tgatctatga tgtggataga aatcaaatgt
     5761 ccaaattgaa aattgaaaac ttccagaaaa gtgtgttttt gccaaaagag aggctatcac
     5821 cggttatttc tatccaatgg aatcctagag acataggaac gatacttata tcatatgagc
     5881 atattacagt catatattct ttcattgact acaaagtccc gcggatctgc cggtctccct
     5941 atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg
     6001 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6061 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6121 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6181 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6241 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6301 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6361 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6421 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6481 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6541 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6601 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6661 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6721 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6781 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6841 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6901 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6961 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7021 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7081 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7141 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7201 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7261 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7321 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7381 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7441 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7501 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7561 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7621 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7681 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7741 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7801 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7861 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7921 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     7981 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     8041 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     8101 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     8161 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     8221 ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
     8281 acgatttagg tgacactata
//
