LOCUS       sequence-128295-        7884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of 2GFP tag at Atg20 C terminus, use auxotrophic marker 
            URA3(Candida albicans).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7884)
  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 7884)
  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: LD220
FEATURES             Location/Qualifiers
     source          1..7884
                     /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             1938..2651
                     /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             2688..3404
                     /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             2688..3401
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      3425..3612
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3480..3504)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        3687..4030
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      4849..5046
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(5696..5716)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5696..5714)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5735..5751)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5735..5751)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5748..5770)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5759..5775
                     /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(5783..5789,5790..5807,5808..5813))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5828..5849
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5966..5983)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6137..6725)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6217..6236)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6896..7687,7688..7756))
                     /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     7519..7538
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7757..7861)
                     /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 ccgcggagta tttgctgccc tcagaaggtt ctggttcagt agatctatca ttgtctgtga
      721 tacatgcgtc ggtcaataat agtgatgaaa agttaatacg gcataggata agaatgctca
      781 ctgagttctt gaacaaactt ttgacgaacg aagaaatcac aaaaacatct ataattaccg
      841 attttttgga tcctaataac cataattggc atgaatttgt taacagttcg tccacttttt
      901 catctttacc gaagagcatc ctccagtgta atccactaga tcccactaac accactagaa
      961 ttcatgccat gttaccaata ccgggctcct cgtcacagtt attgttgaat aaggagtcca
     1021 acgacaaaaa aatggataag gaaagaagta agtcgtttac taatattgaa caggattaca
     1081 agcaatatga gaacttgtta gataacggga tttataagta taatagacga accacgaaga
     1141 cttatcacga tttgaaatcg gattacaatg aaatcggtga ggtatttgca cagtttgccc
     1201 atgaacaggc tcaagttggg gaattggccg aacagctttc ttacttaagc aatgcctttt
     1261 cggggtcctc gatttcttta gaaaaattag ttggaagact atactacaat ataaatgagc
     1321 ctttaaatga atctgttcat atggcaactt ctgcaagaga attgatcaaa tacagaaaat
     1381 tgaaatacct gcaaaatgaa atgattaaaa agtctctgaa ttccaaaaga gcacagttgg
     1441 agaagctgga agcgcaaaac aatgaatata aggatgttga caaaataatt gacaacgaaa
     1501 tgtctaaaag tcatactatt aatctggaac gccctaataa taatacaggt agcggtggga
     1561 aatcatatgg tggcaaacta ttcaatggat ttaacaaatt ggcatcaatg gtaaaagatt
     1621 ctgtgaagta tcaagaaaca gatccgcata ccgcaagtat caatttgaag aaagaaattg
     1681 aacaactgtc tgaatcattg gaagttactg agaatgattt ggaggtcatt tcaaaagtca
     1741 tcaaaaatga ccagctacct aaattttcta aagaaaggga agtagattta tcagaaattt
     1801 taaagcatta ttccagatat atgagaaatt atgcccgtca aaatttagaa atttggaaag
     1861 aagtcaaaag acatcaagat tttgcagctt cgtacgctgc aggtcgacgg atcggtgacg
     1921 gtgctggttt aattaacatg tctaaaggtg aagaattatt cactggtgtt gtcccaattt
     1981 tggttgaatt agatggtgat gttaatggtc acaaattttc tgtctccggt gaaggtgaag
     2041 gtgatgctac ttacggtaaa ttgaccttaa aatttatttg tactactggt aaattgccag
     2101 ttccatggcc aaccttagtc actactttaa cttatggtgt tcaatgtttt tctagatacc
     2161 cagatcatat gaaacaacat gactttttca agtctgccat gccagaaggt tatgttcaag
     2221 aaagaactat ttttttcaaa gatgacggta actacaagac cagagctgaa gtcaagtttg
     2281 aaggtgatac cttagttaat agaatcgaat taaaaggtat tgattttaaa gaagatggta
     2341 acattttagg tcacaaattg gaatacaact ataactctca caatgtttac atcatggctg
     2401 acaaacaaaa gaatggtatc aaagttaact tcaaaattag acacaacatt gaagatggtt
     2461 ctgttcaatt agctgaccat tatcaacaaa atactccaat tggtgatggt ccagtcttgt
     2521 taccagacaa ccattactta tccactcaat ctgccttatc caaagatcca aacgaaaaga
     2581 gagaccacat ggtcttgtta gaatttgtta ctgctgctgg tattacccat ggtattgatg
     2641 aattgtacaa aattccagtt ccaacgcgtg atctagagaa gccattcatg tctaaaggtg
     2701 aagaattatt cactggtgtt gtcccaattt tggttgaatt agatggtgat gttaatggtc
     2761 acaaattttc tgtctccggt gaaggtgaag gtgatgctac ttacggtaaa ttgaccttaa
     2821 aatttatttg tactactggt aaattgccag ttccatggcc aaccttagtc actactttaa
     2881 cttatggtgt tcaatgtttt tctagatacc cagatcatat gaaacaacat gactttttca
     2941 agtctgccat gccagaaggt tatgttcaag aaagaactat ttttttcaaa gatgacggta
     3001 actacaagac cagagctgaa gtcaagtttg aaggtgatac cttagttaat agaatcgaat
     3061 taaaaggtat tgattttaaa gaagatggta acattttagg tcacaaattg gaatacaact
     3121 ataactctca caatgtttac atcatggctg acaaacaaaa gaatggtatc aaagttaact
     3181 tcaaaattag acacaacatt gaagatggtt ctgttcaatt agctgaccat tatcaacaaa
     3241 atactccaat tggtgatggt ccagtcttgt taccagacaa ccattactta tccactcaat
     3301 ctgccttatc caaagatcca aacgaaaaga gagaccacat ggtcttgtta gaatttgtta
     3361 ctgctgctgg tattacccat ggtatggatg aattatataa ataaggcgcg ccacttctaa
     3421 ataagcgaat ttcttatgat ttatgatttt tattattaaa ttagttataa aaaaaataag
     3481 tgtatacaaa ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa
     3541 ctctttcctg taggtcaggt tgctttctca ggtatagtat gaggtcgctc ttattgacca
     3601 cacctctacc ggcagatccg ctagggataa cagggtaata tagatctgtt tagcttgcct
     3661 cgtccccgcc gggtcacccg gccagcgaca tggaggccca gaataccctc cttgacagtc
     3721 ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag cccatacatc
     3781 cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga agcaaaaatt
     3841 acggctcctc gctgcagacc tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt
     3901 gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca ctgaggttct
     3961 tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc acatccgaac
     4021 ataaacaacc atgacagtca acactaagac ctatagtgag agagcagaaa ctcatgcctc
     4081 accagtagca cagcgattat ttcgattaat ggaactgaag aaaaccaatt tatgtgcatc
     4141 aattgacgtt gataccacta aggaattcct tgaattaatt gataaattag gtccttatgt
     4201 atgcttaatc aagactcata ttgatataat caatgatttt tcctatgaat ccactattga
     4261 accattatta gaactttcac gtaaacatca atttatgatt tttgaagata gaaaatttgc
     4321 tgatattggt aataccgtaa agaaacaata tattggtgga gtttataaaa ttagtagttg
     4381 ggcagatatt accaatgctc atggtgtcac tgggaatgga gtggttgaag gattaaaaca
     4441 gggagctaaa gaaaccacca ccaaccaaga gccaagaggg ttattgatgt tagctgaatt
     4501 atcatcagtg ggatcattag catatggaga atattctcaa aaaactgttg aaattgctaa
     4561 atccgataag gaatttgtta ttggatttat tgcccaacgt gatatgggtg gccaagaaga
     4621 aggatttgat tggcttatta tgacacctgg agttggatta gatgataaag gtgatggatt
     4681 aggacaacaa tatagaactg ttgatgaagt tgttagcact ggaactgata ttatcattgt
     4741 tggtagagga ttgtttggta aaggaagaga tccagatatt gaaggtaaaa ggtatagaaa
     4801 tgctggttgg aatgcttatt tgaaaaagac tggccaatta taatcagtac tgacaataaa
     4861 aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta
     4921 ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat
     4981 gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct
     5041 atactgctgt cgattcgata ctaacgccgc catccagtgt cgaaaacgag ctcgaattca
     5101 tcgatgatat cagatccact agtggcctat gctagtatta tgagcatttt tttttagcgt
     5161 tatagcatac ttgatatata tatatataaa caacgtgaag atcaaataac gtcaaatgta
     5221 ttaaatagac taaaatatta tgcacttctc ttcggattta ttgctcgcgg gctggttgac
     5281 gttacatggt ccattccact gaaatgtttt aagagatgcc tatatggcca actcaaagtt
     5341 ttgaaaaagg gaactaggtt cacataatat ggggcattta gtgtttgcag atgattatgc
     5401 tccatcgcag agattatcgc tagtgccagt tgggaaggta tgatgtcagg agccaataac
     5461 ttagatggtg tagggacatc aataaacata ttagttttga tttttcccgg gcacaccagt
     5521 agtgttttta ttccagtttt attacattcc gtggacaaac ttctgaaatg cctgctcata
     5581 cacttgtgaa aaccaatcat tgctcctttt gatgcaccat acgatgtcag ccttgcagga
     5641 gtcagttctc cgaggactga agctcgaggg ggggcccggt acccagcttt tgttcccttt
     5701 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5761 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5821 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5881 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5941 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6001 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6061 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6121 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6181 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6241 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6301 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6361 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6421 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6481 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6541 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     6601 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6661 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6721 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6781 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6841 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6901 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6961 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7021 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7081 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7141 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7201 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7261 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7321 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7381 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7441 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7501 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7561 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7621 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7681 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7741 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7801 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7861 gcacatttcc ccgaaaagtg ccac
//
