LOCUS       sequence-128298-        8784 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of 2GFP tag at Atg24 C terminus, use auxotrophic marker 
            URA3(Candida albicans).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8784)
  AUTHORS   Li D, Song JZ, Shan MH, Li SP, Liu W, Li H, Zhu J, Wang Y, Lin J, 
            Xie Z
  TITLE     A fluorescent tool set for yeast Atg proteins.
  JOURNAL   Autophagy. 2015;11(6):954-60. doi: 10.1080/15548627.2015.1040971.
  PUBMED    25998947
REFERENCE   2  (bases 1 to 8784)
  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: 
            "Autophagy."; date: "2015"; volume: "11(6):954-60. doi"; pages: " 
            10.1080/15548627.2015.1040971"
COMMENT     Sequence Label: LD224
FEATURES             Location/Qualifiers
     source          1..8784
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2487..3200
                     /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             3237..3953
                     /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             3237..3950
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      3974..4161
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4029..4053)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        4236..4579
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      5398..5595
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(6596..6616)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6596..6614)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6635..6651)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6635..6651)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6648..6670)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6659..6675
                     /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)."
     promoter        complement(join(6683..6689,6690..6707,6708..6713))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6728..6749
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6866..6883)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7037..7625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7117..7136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7796..8587,8588..8656))
                     /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     8419..8438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8657..8761)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtttc tgctgaaagc ccaagctttc ataaattaat aggtttcgtg gtctagtcgg
      721 ttatggcatc tgcttaacac gcagaacgtc cccagttcga tcctgggcga aatcaatttt
      781 ttgttttttt aattcacaag aataacctgg tattgttata agcaagccac cctttgcgta
      841 cgatcaaaca ccctataaaa tctatatgca atactgagat attgtatttt ttctccttta
      901 attgtttata gttcagaata acagttcaaa aatctatgcc gcgctcacag ggtcctgccg
      961 ggtattcgcc ctccagcttg aaaggggtgg caatgcccaa gagcgatgta tagtttagta
     1021 tcaaggaatt gagagaggta ttacaccaag gatttccgtg aacgaagtga cgacgcaaga
     1081 ttaacagatt aaattctggt agtaggctaa gccgttatta aactttattt acggtatacc
     1141 acaatactgc tctttttgtt gaggatatga cagacaaagg caagaacgat ttaacaagca
     1201 aagctaagga caaagccaga ggcaaccccg agaagccacc atattggttt gaaatcattg
     1261 tttctgatcc acaaaaaaga actggtgacc caggctcttc atctggctac gtgagttatc
     1321 agatatcaac aaagactaac aacacgtcat tttatgataa tcgtggcgat ccagaatcta
     1381 ttatagttgt acatagaaga tacagtgacc tattgctcct gcatgatatt ttactcaaca
     1441 ggttccccac ctgcatcatc ccgccattac ctgataagaa agtttttcaa tacatagcag
     1501 gcgataggtt tagccaaaga tttacacaaa agagatgtca tagtttgcaa aatttcctaa
     1561 gaagggtctc tttgcatccc gatctttctc aatcaaaagt cttcaaaact tttttggtga
     1621 gtaaagattg ggaatctcac cgcaaagtcc tccaggatag cttgcaacca aataaagatg
     1681 aagttactga tgcttttatg aatgccttca aaacagttca caagcaaaac gaagaattca
     1741 ctgagataag ggagaaaagt gataaactag atcggactgt gacaaagata gataaacttt
     1801 ttcataaggt tgtgaaaaaa aatgattcga tgtctgaaga ctatacaaag ttgggcagta
     1861 atttacagga attacaagaa ttggtcacag gcgaaaacga ggaacttgct gcgaagttga
     1921 agatttttaa tgaaggtgta acacagctgt catatggctt gcaagatctt acaaagtatc
     1981 tggattacga atatattgtt gatttgaagg atcttgaaca ctacattgat agtatgcgac
     2041 agctgatcaa attgaaagac cagaaacaga tagactacga agaactaagt gattatttaa
     2101 ccaggtcaat caaagaaaaa aataacttga tttctggata cggaggtagt aatttctttg
     2161 ccaataaatt ggaagaacta gcgggaataa atcaggaggc ttcacgtaga gaaaaaatca
     2221 ataaactaga aggcaaaata acgtcattaa caggtgaatt agaaaacgct aaaaaggtgg
     2281 cggatggatt cgaacaggaa tgcttgaagg aaatagacca tttcgaaagt gtaaaaacgg
     2341 ctgaaatcaa gaaatctctg ggctcactag cggaccatca tatcgaattc tacgaaagga
     2401 tacttgaagc atgggaaaaa gtagacgata gtctagcttc gtacgctgca ggtcgacgga
     2461 tcggtgacgg tgctggttta attaacatgt ctaaaggtga agaattattc actggtgttg
     2521 tcccaatttt ggttgaatta gatggtgatg ttaatggtca caaattttct gtctccggtg
     2581 aaggtgaagg tgatgctact tacggtaaat tgaccttaaa atttatttgt actactggta
     2641 aattgccagt tccatggcca accttagtca ctactttaac ttatggtgtt caatgttttt
     2701 ctagataccc agatcatatg aaacaacatg actttttcaa gtctgccatg ccagaaggtt
     2761 atgttcaaga aagaactatt tttttcaaag atgacggtaa ctacaagacc agagctgaag
     2821 tcaagtttga aggtgatacc ttagttaata gaatcgaatt aaaaggtatt gattttaaag
     2881 aagatggtaa cattttaggt cacaaattgg aatacaacta taactctcac aatgtttaca
     2941 tcatggctga caaacaaaag aatggtatca aagttaactt caaaattaga cacaacattg
     3001 aagatggttc tgttcaatta gctgaccatt atcaacaaaa tactccaatt ggtgatggtc
     3061 cagtcttgtt accagacaac cattacttat ccactcaatc tgccttatcc aaagatccaa
     3121 acgaaaagag agaccacatg gtcttgttag aatttgttac tgctgctggt attacccatg
     3181 gtattgatga attgtacaaa attccagttc caacgcgtga tctagagaag ccattcatgt
     3241 ctaaaggtga agaattattc actggtgttg tcccaatttt ggttgaatta gatggtgatg
     3301 ttaatggtca caaattttct gtctccggtg aaggtgaagg tgatgctact tacggtaaat
     3361 tgaccttaaa atttatttgt actactggta aattgccagt tccatggcca accttagtca
     3421 ctactttaac ttatggtgtt caatgttttt ctagataccc agatcatatg aaacaacatg
     3481 actttttcaa gtctgccatg ccagaaggtt atgttcaaga aagaactatt tttttcaaag
     3541 atgacggtaa ctacaagacc agagctgaag tcaagtttga aggtgatacc ttagttaata
     3601 gaatcgaatt aaaaggtatt gattttaaag aagatggtaa cattttaggt cacaaattgg
     3661 aatacaacta taactctcac aatgtttaca tcatggctga caaacaaaag aatggtatca
     3721 aagttaactt caaaattaga cacaacattg aagatggttc tgttcaatta gctgaccatt
     3781 atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt accagacaac cattacttat
     3841 ccactcaatc tgccttatcc aaagatccaa acgaaaagag agaccacatg gtcttgttag
     3901 aatttgttac tgctgctggt attacccatg gtatggatga attatataaa taaggcgcgc
     3961 cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa
     4021 aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt
     4081 cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagtatg aggtcgctct
     4141 tattgaccac acctctaccg gcagatccgc tagggataac agggtaatat agatctgttt
     4201 agcttgcctc gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc
     4261 ttgacagtct tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc
     4321 ccatacatcc ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa
     4381 gcaaaaatta cggctcctcg ctgcagacct gcgagcaggg aaacgctccc ctcacagacg
     4441 cgttgaattg tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac
     4501 tgaggttctt ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca
     4561 catccgaaca taaacaacca tgacagtcaa cactaagacc tatagtgaga gagcagaaac
     4621 tcatgcctca ccagtagcac agcgattatt tcgattaatg gaactgaaga aaaccaattt
     4681 atgtgcatca attgacgttg ataccactaa ggaattcctt gaattaattg ataaattagg
     4741 tccttatgta tgcttaatca agactcatat tgatataatc aatgattttt cctatgaatc
     4801 cactattgaa ccattattag aactttcacg taaacatcaa tttatgattt ttgaagatag
     4861 aaaatttgct gatattggta ataccgtaaa gaaacaatat attggtggag tttataaaat
     4921 tagtagttgg gcagatatta ccaatgctca tggtgtcact gggaatggag tggttgaagg
     4981 attaaaacag ggagctaaag aaaccaccac caaccaagag ccaagagggt tattgatgtt
     5041 agctgaatta tcatcagtgg gatcattagc atatggagaa tattctcaaa aaactgttga
     5101 aattgctaaa tccgataagg aatttgttat tggatttatt gcccaacgtg atatgggtgg
     5161 ccaagaagaa ggatttgatt ggcttattat gacacctgga gttggattag atgataaagg
     5221 tgatggatta ggacaacaat atagaactgt tgatgaagtt gttagcactg gaactgatat
     5281 tatcattgtt ggtagaggat tgtttggtaa aggaagagat ccagatattg aaggtaaaag
     5341 gtatagaaat gctggttgga atgcttattt gaaaaagact ggccaattat aatcagtact
     5401 gacaataaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt ttatattgta
     5461 gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct cgacatcatc
     5521 tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg
     5581 ctggtcgcta tactgctgtc gattcgatac taacgccgcc atccagtgtc gaaaacgagc
     5641 tcgaattcat cgatgatatc agatccacta gtggcctatg ctagtggtgc tcttaatgtt
     5701 ttcccattac tactgataat accttgggca ctacgattat ctgatcacat ttaacagaga
     5761 aatttaaaaa aagaaaaata tctatataaa aatatgtaaa tgttgctatt attttatgat
     5821 gttctgattt ttttcgtcaa accttagatt tcttatgtac attaaactta aacattttgc
     5881 attgttcttc cagttcttgg attatattat cttggtggga gtcaatcaaa tcccaatcga
     5941 cgctattact ggacaagtca actttattcg caaaacaagt cctgtaatca tcaccaaaag
     6001 tctcaatgaa tgcttcttca ttaagatact ttgaccattc cataggcgga aacgtgtttt
     6061 tatccaacac tctgtcactc ttggaccgtg gctttggcgc cctttcattt tgtcttacat
     6121 aaaagtatct cagcagcatt attgcttctt tcctcaagat ccccggtata ctctcgtacc
     6181 ctgccgcact attgttcttg ggcactaatg tacacgtatc atgatttact gacaagacag
     6241 taccgttgcc tccaaatctc tcgttgccac aaccgaacac cacctttcca atgtctaatt
     6301 gcttgagagc agatgcacac attatacacg gttctacagt aacatataga gtaatgtctt
     6361 tgaacacgtc aacaactcct cgggagccca acatcgcctt gatctgatcg atccccatga
     6421 actcagcatg tgctactcca gttaatgact tgttcgtatc gttcatgcca taagccatca
     6481 cctggccagt tggcgtatgt acaaagatgc aagctaccgg tgtctcatcg tggtcaagag
     6541 cgtatctagc caaacggaca gcctcgaggg ggggcccggt acccagcttt tgttcccttt
     6601 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     6661 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     6721 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     6781 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6841 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6901 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6961 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     7021 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     7081 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     7141 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     7201 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     7261 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     7321 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     7381 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     7441 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     7501 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7561 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7621 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7681 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7741 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7801 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7861 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7921 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7981 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     8041 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     8101 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     8161 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     8221 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     8281 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     8341 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     8401 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     8461 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     8521 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     8581 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     8641 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     8701 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     8761 gcacatttcc ccgaaaagtg ccac
//
