LOCUS       sequence-128300-        7434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of 2GFP tag at Atg31 C terminus, use auxotrophic marker 
            URA3(Candida albicans).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7434)
  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 7434)
  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: LD231
FEATURES             Location/Qualifiers
     source          1..7434
                     /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             1506..2219
                     /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             2256..2972
                     /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             2256..2969
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2993..3180
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3048..3072)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        3255..3598
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      4417..4614
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(5246..5266)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5246..5264)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5285..5301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5285..5301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5298..5320)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5309..5325
                     /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(5333..5339,5340..5357,5358..5363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5378..5399
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5516..5533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5687..6275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5767..5786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6446..7237,7238..7306))
                     /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     7069..7088
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7307..7411)
                     /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 ccgcggggtc cagtggttca agacgtcgcc tttacacggc gaagatcccg agttcgaacc
      721 tcggttggat cagaatcttt tttatcgtaa aagataataa tttgaagtac atatccataa
      781 tataagggtg gtcaatatcc aagataaacg tcgcattccc attttcttat taagccggta
      841 aacattgctg aaatctgcga acaggaatga atgttacagt tactgtttat gataaaaatg
      901 tcaagtacag attagaagag aatatcaaaa ataacaaggg tcccagcaac gatgatcaac
      961 cagcatacaa taatgaatca aagagcacgg acggaagtga ttatgcaatg tttcccacta
     1021 acattaagta catatttgag gacaacaatg atgaactagt agattcaagt gatgctgctt
     1081 tgacagcagg aatagataag gtcggagatg aacttgaaaa tgtcataatt gtccaattgg
     1141 atgaatcggg atctcttgag gatatcacac taatcagcga ccaatatgag cttttgtcac
     1201 ataggacaaa tagtctcagc ttggaggaaa accaaatgag aacattatca tcacatggtg
     1261 atgataaatc taatgatgaa gaggaagaac ttagcgttga cagtgataga ttcagagtgg
     1321 actcggacat tgagctcgat gtaatttcac aattttgcga tctgtctcct tttcttcgcg
     1381 atttgagttt gaatgactta attaaactct acgtcacaca aaatgaacaa ttacaaatgc
     1441 tctccaattc cgtagcttcg tacgctgcag gtcgacggat cggtgacggt gctggtttaa
     1501 ttaacatgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg gttgaattag
     1561 atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt gatgctactt
     1621 acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt ccatggccaa
     1681 ccttagtcac tactttaact tatggtgttc aatgtttttc tagataccca gatcatatga
     1741 aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa agaactattt
     1801 ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa ggtgatacct
     1861 tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac attttaggtc
     1921 acaaattgga atacaactat aactctcaca atgtttacat catggctgac aaacaaaaga
     1981 atggtatcaa agttaacttc aaaattagac acaacattga agatggttct gttcaattag
     2041 ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta ccagacaacc
     2101 attacttatc cactcaatct gccttatcca aagatccaaa cgaaaagaga gaccacatgg
     2161 tcttgttaga atttgttact gctgctggta ttacccatgg tattgatgaa ttgtacaaaa
     2221 ttccagttcc aacgcgtgat ctagagaagc cattcatgtc taaaggtgaa gaattattca
     2281 ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac aaattttctg
     2341 tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa tttatttgta
     2401 ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaact tatggtgttc
     2461 aatgtttttc tagataccca gatcatatga aacaacatga ctttttcaag tctgccatgc
     2521 cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac tacaagacca
     2581 gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta aaaggtattg
     2641 attttaaaga agatggtaac attttaggtc acaaattgga atacaactat aactctcaca
     2701 atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc aaaattagac
     2761 acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat actccaattg
     2821 gtgatggtcc agtcttgtta ccagacaacc attacttatc cactcaatct gccttatcca
     2881 aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact gctgctggta
     2941 ttacccatgg tatggatgaa ttatataaat aaggcgcgcc acttctaaat aagcgaattt
     3001 cttatgattt atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt
     3061 ttaaagtgac tcttaggttt taaaacgaaa attcttattc ttgagtaact ctttcctgta
     3121 ggtcaggttg ctttctcagg tatagtatga ggtcgctctt attgaccaca cctctaccgg
     3181 cagatccgct agggataaca gggtaatata gatctgttta gcttgcctcg tccccgccgg
     3241 gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca
     3301 gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat
     3361 catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc
     3421 tgcagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg
     3481 cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac
     3541 ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat
     3601 gacagtcaac actaagacct atagtgagag agcagaaact catgcctcac cagtagcaca
     3661 gcgattattt cgattaatgg aactgaagaa aaccaattta tgtgcatcaa ttgacgttga
     3721 taccactaag gaattccttg aattaattga taaattaggt ccttatgtat gcttaatcaa
     3781 gactcatatt gatataatca atgatttttc ctatgaatcc actattgaac cattattaga
     3841 actttcacgt aaacatcaat ttatgatttt tgaagataga aaatttgctg atattggtaa
     3901 taccgtaaag aaacaatata ttggtggagt ttataaaatt agtagttggg cagatattac
     3961 caatgctcat ggtgtcactg ggaatggagt ggttgaagga ttaaaacagg gagctaaaga
     4021 aaccaccacc aaccaagagc caagagggtt attgatgtta gctgaattat catcagtggg
     4081 atcattagca tatggagaat attctcaaaa aactgttgaa attgctaaat ccgataagga
     4141 atttgttatt ggatttattg cccaacgtga tatgggtggc caagaagaag gatttgattg
     4201 gcttattatg acacctggag ttggattaga tgataaaggt gatggattag gacaacaata
     4261 tagaactgtt gatgaagttg ttagcactgg aactgatatt atcattgttg gtagaggatt
     4321 gtttggtaaa ggaagagatc cagatattga aggtaaaagg tatagaaatg ctggttggaa
     4381 tgcttatttg aaaaagactg gccaattata atcagtactg acaataaaaa gattcttgtt
     4441 ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat
     4501 gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt
     4561 gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg
     4621 attcgatact aacgccgcca tccagtgtcg aaaacgagct cgaattcatc gatgatatca
     4681 gatccactag tggcctatgc tgaaagcaaa aatgaagccg catggatgag actctcttaa
     4741 cttgtttatg taggtatatg ttctctatat tttatgataa ttctgcaaaa tttgctggca
     4801 tcggatttat tttgatagaa tagaattttt caatttctct aagttttgct aaatcagctt
     4861 tggtaatgaa gtttatagcc acaccctttc taccaaatcg accacttcta ccgatacgat
     4921 ggatgtaatt ttctattatt tctggtaggt catagttaat gactaaagaa acttgttgga
     4981 catcaatacc gcgcgcccaa acatccgtcg atatcagtac acgagaatga cctgttctaa
     5041 aatcattcat tactttatct ctttcttctt gtttcatatc accatgcatg gagactacag
     5101 caaagtttga ttggattagc cgctgagata accaatctac ttttttcttg gtgttacaga
     5161 atatcacaca ttgtgtaatg gtcaaagaat cgtatatatc acacagagta tcctcgaggg
     5221 ggggcccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     5281 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5341 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5401 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5461 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5521 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5581 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5641 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5701 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5761 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5821 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5881 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5941 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6001 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6061 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6121 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6181 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6241 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6301 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6361 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6421 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6481 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6541 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6601 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6661 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6721 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6781 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6841 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6901 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6961 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7021 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7081 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7141 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7201 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7261 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7321 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7381 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccac
//
