LOCUS       sequence-128291-        7935 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of 2GFP tag at Atg17 C terminus, use auxotrophic marker 
            URA3(Candida albicans).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7935)
  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 7935)
  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: LD217
FEATURES             Location/Qualifiers
     source          1..7935
                     /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"
     RBS             1413..1424
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1669..2382
                     /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             2419..3135
                     /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             2419..3132
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      3156..3343
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3211..3235)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        3418..3761
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      4580..4777
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(5701..5717)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5702..5718)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(5747..5767)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5747..5765)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5786..5802)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5786..5802)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5799..5821)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5810..5826
                     /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(5834..5840,5841..5858,5859..5864))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5879..5900
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6017..6034)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6188..6776)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6268..6287)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6947..7738,7739..7807))
                     /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     7570..7589
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7808..7912)
                     /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 ccgcggaaag tacaaaggtt ggatggtata gtaaatgaat tgagcatatc agaaaaagat
      721 gataccgatc cctctaaact aggagattac atctcgagag acaacgttaa cttattgaat
      781 gacaaactga aagaagtgcc agtaattgag cgtcaaattg aaaacattaa gcttcaatac
      841 gaaaatatgg tcagaaaagt taacaaagaa ctgattgata ccaagttgac ggacgtaact
      901 caaaaattcc aaagtaaatt tggtatagat aacctgatgg aaacaaatgt tgcagagcag
      961 tttagcaggg aactaacgga ccttgaaaaa gatttagcag agataatgaa ttcattgaca
     1021 cagcactttg ataagacatt gctgttacaa gataaaaaaa ttgataacga tgaacgtgag
     1081 gagctgttta aggtggtaca aggcgacgac aaagaactat acaacatttt caaaactctg
     1141 catgaggtaa ttgatgacgt ggacaaaaca attcttaact tgggtcaatt tttgcaggca
     1201 aaaataaagg aaaagacaga actacacagc gaagtttctg aaataataaa cgatttcaat
     1261 agaaatttgg aatatctatt aatctttaaa gatatttcca atctgattga tagcttcaaa
     1321 aattcctgta cacaagatat tcaaacaact aaggaacttt gtgaatttta tgataatttt
     1381 gaagaaagct acggtaactt agttctagaa gcaaagagga gaaaggatgt ggcaaacaga
     1441 atgaaaacta tattgaaaga ttgtgaaaag cagttgcaga atttagatgc tcaggaccag
     1501 gaagaacgtc agaattttat agcggaaaat ggaacttatc ttcctgagac aatctggccc
     1561 ggtaaaattg acgatttttc ttccctgtac actttaaatt acaacgtgaa gaatcctgct
     1621 tcgtacgctg caggtcgacg gatcggtgac ggtgctggtt taattaacat gtctaaaggt
     1681 gaagaattat tcactggtgt tgtcccaatt ttggttgaat tagatggtga tgttaatggt
     1741 cacaaatttt ctgtctccgg tgaaggtgaa ggtgatgcta cttacggtaa attgacctta
     1801 aaatttattt gtactactgg taaattgcca gttccatggc caaccttagt cactacttta
     1861 acttatggtg ttcaatgttt ttctagatac ccagatcata tgaaacaaca tgactttttc
     1921 aagtctgcca tgccagaagg ttatgttcaa gaaagaacta tttttttcaa agatgacggt
     1981 aactacaaga ccagagctga agtcaagttt gaaggtgata ccttagttaa tagaatcgaa
     2041 ttaaaaggta ttgattttaa agaagatggt aacattttag gtcacaaatt ggaatacaac
     2101 tataactctc acaatgttta catcatggct gacaaacaaa agaatggtat caaagttaac
     2161 ttcaaaatta gacacaacat tgaagatggt tctgttcaat tagctgacca ttatcaacaa
     2221 aatactccaa ttggtgatgg tccagtcttg ttaccagaca accattactt atccactcaa
     2281 tctgccttat ccaaagatcc aaacgaaaag agagaccaca tggtcttgtt agaatttgtt
     2341 actgctgctg gtattaccca tggtattgat gaattgtaca aaattccagt tccaacgcgt
     2401 gatctagaga agccattcat gtctaaaggt gaagaattat tcactggtgt tgtcccaatt
     2461 ttggttgaat tagatggtga tgttaatggt cacaaatttt ctgtctccgg tgaaggtgaa
     2521 ggtgatgcta cttacggtaa attgacctta aaatttattt gtactactgg taaattgcca
     2581 gttccatggc caaccttagt cactacttta acttatggtg ttcaatgttt ttctagatac
     2641 ccagatcata tgaaacaaca tgactttttc aagtctgcca tgccagaagg ttatgttcaa
     2701 gaaagaacta tttttttcaa agatgacggt aactacaaga ccagagctga agtcaagttt
     2761 gaaggtgata ccttagttaa tagaatcgaa ttaaaaggta ttgattttaa agaagatggt
     2821 aacattttag gtcacaaatt ggaatacaac tataactctc acaatgttta catcatggct
     2881 gacaaacaaa agaatggtat caaagttaac ttcaaaatta gacacaacat tgaagatggt
     2941 tctgttcaat tagctgacca ttatcaacaa aatactccaa ttggtgatgg tccagtcttg
     3001 ttaccagaca accattactt atccactcaa tctgccttat ccaaagatcc aaacgaaaag
     3061 agagaccaca tggtcttgtt agaatttgtt actgctgctg gtattaccca tggtatggat
     3121 gaattatata aataaggcgc gccacttcta aataagcgaa tttcttatga tttatgattt
     3181 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg
     3241 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc
     3301 aggtatagta tgaggtcgct cttattgacc acacctctac cggcagatcc gctagggata
     3361 acagggtaat atagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac
     3421 atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt
     3481 gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat
     3541 tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag
     3601 ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat
     3661 ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg
     3721 ctaggataca gttctcacat cacatccgaa cataaacaac catgacagtc aacactaaga
     3781 cctatagtga gagagcagaa actcatgcct caccagtagc acagcgatta tttcgattaa
     3841 tggaactgaa gaaaaccaat ttatgtgcat caattgacgt tgataccact aaggaattcc
     3901 ttgaattaat tgataaatta ggtccttatg tatgcttaat caagactcat attgatataa
     3961 tcaatgattt ttcctatgaa tccactattg aaccattatt agaactttca cgtaaacatc
     4021 aatttatgat ttttgaagat agaaaatttg ctgatattgg taataccgta aagaaacaat
     4081 atattggtgg agtttataaa attagtagtt gggcagatat taccaatgct catggtgtca
     4141 ctgggaatgg agtggttgaa ggattaaaac agggagctaa agaaaccacc accaaccaag
     4201 agccaagagg gttattgatg ttagctgaat tatcatcagt gggatcatta gcatatggag
     4261 aatattctca aaaaactgtt gaaattgcta aatccgataa ggaatttgtt attggattta
     4321 ttgcccaacg tgatatgggt ggccaagaag aaggatttga ttggcttatt atgacacctg
     4381 gagttggatt agatgataaa ggtgatggat taggacaaca atatagaact gttgatgaag
     4441 ttgttagcac tggaactgat attatcattg ttggtagagg attgtttggt aaaggaagag
     4501 atccagatat tgaaggtaaa aggtatagaa atgctggttg gaatgcttat ttgaaaaaga
     4561 ctggccaatt ataatcagta ctgacaataa aaagattctt gttttcaaga acttgtcatt
     4621 tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg tgatttatat
     4681 tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga aagtaatatc
     4741 atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat actaacgccg
     4801 ccatccagtg tcgaaaacga gctcgaattc atcgatgata tcagatccac tagtggccta
     4861 tgctagaaaa aaggaaaaaa aggatacggt acaaagattc aataattgta tcaatatttc
     4921 gaaaaatact ttatcaaaaa agcaatactt gaaggagaaa ggaaaaaatt cgctgtcttc
     4981 ttcactttta cgacaaagaa aaatgtgccc ttttcaacta aaagtcatga atgaataaat
     5041 aaatgattac atatgcgtat attttatagt gaacaacttc tgacatatta tcccgaatgt
     5101 gagtaatcat gcttgtgctg tgaatctgtg ttggtgtttg gctgtgaccc tgccaaaaag
     5161 gtgctcgaag tgccggaaac aattccactt ttatccaaac tgctcgtagt ggacgagtgg
     5221 gataacgttc ttgttaaata ttttccaaaa agtaaactgt ttgggccaaa tgagtcgcta
     5281 tgttctaata caaattggct atgattggac cctgaggtgt gatgatccct tccagaaaag
     5341 cttgcctttt tattggggtt cttgtgcgaa attattcgag cagtaatcat gttattcctg
     5401 ctacgattgg cctcactgag agtacgacta tatctttcta tctcctcagc gaaattttcc
     5461 tcaaaaagac gcactagaag atcatgtgat ggttttagat cctttgatgg tagtagttct
     5521 gcatgcaacg ttagacagcg acctaatact gcaacaagct catcaaaagc tagcgtcaat
     5581 cttcctaaat catcagtact gaactgcttg gatgtggagg gttctaaaaa ggccttgtac
     5641 tgtgatatgc ctccattaac tgggatcccc cgggctgcag gaattcgata tcaagcttat
     5701 cgataccgtc gacctcgagg gggggcccgg tacccagctt ttgttccctt tagtgagggt
     5761 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5821 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5881 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5941 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6001 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     6061 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6121 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6181 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6241 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6301 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6361 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6421 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6481 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6541 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6601 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     6661 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6721 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6781 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6841 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6901 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6961 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     7021 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     7081 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7141 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7201 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7261 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7321 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7381 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7441 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7501 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7561 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7621 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7681 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7741 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7801 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7861 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7921 cccgaaaagt gccac
//
