LOCUS       sequence-128296-        7625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of 2GFP tag at Atg21 C terminus, use auxotrophic marker 
            URA3(Candida albicans).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7625)
  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 7625)
  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: LD221
FEATURES             Location/Qualifiers
     source          1..7625
                     /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             892..903
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             1206..1919
                     /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             1956..2672
                     /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             1956..2669
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2693..2880
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2748..2772)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2955..3298
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      4117..4314
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(5437..5457)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5437..5455)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5476..5492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5476..5492)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5489..5511)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5500..5516
                     /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(5524..5530,5531..5548,5549..5554))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5569..5590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5707..5724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5878..6466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5958..5977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6637..7428,7429..7497))
                     /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     7260..7279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7498..7602)
                     /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 ccgcggcttc aaggtcactg tttaaagaat ttagacgggg taccagattg tgcaatttgt
      721 atcagctagc tttcgataag agtatgacta tgattggatg tgtaggtgat acggacacta
      781 ttcatctttt caaacttgat gacgcctcta atagcctccc tggagataat tctagcaatg
      841 gccactggaa tgaagaggaa tatatactag catcaaattc aaatcccagt atgggaactc
      901 caaaggaaat acctctctcg aaaccaagaa ttgcgaatta cttctcgaaa aaaataaaat
      961 cttcaatacc aaaccagaac ctgagccgta actttgctta tataacagtt aatgaatcga
     1021 acaggagttg cttagggttt ccagacgaat ttcccaacca agtctatatt gcctctgatg
     1081 atggaacctt cagtatatat agtattccgt caaaacctgg tgaatgtgtg ctgactaaaa
     1141 ataataaatt tacagcttcg tacgctgcag gtcgacggat cggtgacggt gctggtttaa
     1201 ttaacatgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg gttgaattag
     1261 atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt gatgctactt
     1321 acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt ccatggccaa
     1381 ccttagtcac tactttaact tatggtgttc aatgtttttc tagataccca gatcatatga
     1441 aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa agaactattt
     1501 ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa ggtgatacct
     1561 tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac attttaggtc
     1621 acaaattgga atacaactat aactctcaca atgtttacat catggctgac aaacaaaaga
     1681 atggtatcaa agttaacttc aaaattagac acaacattga agatggttct gttcaattag
     1741 ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta ccagacaacc
     1801 attacttatc cactcaatct gccttatcca aagatccaaa cgaaaagaga gaccacatgg
     1861 tcttgttaga atttgttact gctgctggta ttacccatgg tattgatgaa ttgtacaaaa
     1921 ttccagttcc aacgcgtgat ctagagaagc cattcatgtc taaaggtgaa gaattattca
     1981 ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac aaattttctg
     2041 tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa tttatttgta
     2101 ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaact tatggtgttc
     2161 aatgtttttc tagataccca gatcatatga aacaacatga ctttttcaag tctgccatgc
     2221 cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac tacaagacca
     2281 gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta aaaggtattg
     2341 attttaaaga agatggtaac attttaggtc acaaattgga atacaactat aactctcaca
     2401 atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc aaaattagac
     2461 acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat actccaattg
     2521 gtgatggtcc agtcttgtta ccagacaacc attacttatc cactcaatct gccttatcca
     2581 aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact gctgctggta
     2641 ttacccatgg tatggatgaa ttatataaat aaggcgcgcc acttctaaat aagcgaattt
     2701 cttatgattt atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt
     2761 ttaaagtgac tcttaggttt taaaacgaaa attcttattc ttgagtaact ctttcctgta
     2821 ggtcaggttg ctttctcagg tatagtatga ggtcgctctt attgaccaca cctctaccgg
     2881 cagatccgct agggataaca gggtaatata gatctgttta gcttgcctcg tccccgccgg
     2941 gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca
     3001 gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat
     3061 catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc
     3121 tgcagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg
     3181 cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac
     3241 ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat
     3301 gacagtcaac actaagacct atagtgagag agcagaaact catgcctcac cagtagcaca
     3361 gcgattattt cgattaatgg aactgaagaa aaccaattta tgtgcatcaa ttgacgttga
     3421 taccactaag gaattccttg aattaattga taaattaggt ccttatgtat gcttaatcaa
     3481 gactcatatt gatataatca atgatttttc ctatgaatcc actattgaac cattattaga
     3541 actttcacgt aaacatcaat ttatgatttt tgaagataga aaatttgctg atattggtaa
     3601 taccgtaaag aaacaatata ttggtggagt ttataaaatt agtagttggg cagatattac
     3661 caatgctcat ggtgtcactg ggaatggagt ggttgaagga ttaaaacagg gagctaaaga
     3721 aaccaccacc aaccaagagc caagagggtt attgatgtta gctgaattat catcagtggg
     3781 atcattagca tatggagaat attctcaaaa aactgttgaa attgctaaat ccgataagga
     3841 atttgttatt ggatttattg cccaacgtga tatgggtggc caagaagaag gatttgattg
     3901 gcttattatg acacctggag ttggattaga tgataaaggt gatggattag gacaacaata
     3961 tagaactgtt gatgaagttg ttagcactgg aactgatatt atcattgttg gtagaggatt
     4021 gtttggtaaa ggaagagatc cagatattga aggtaaaagg tatagaaatg ctggttggaa
     4081 tgcttatttg aaaaagactg gccaattata atcagtactg acaataaaaa gattcttgtt
     4141 ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat
     4201 gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt
     4261 gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg
     4321 attcgatact aacgccgcca tccagtgtcg aaaacgagct cgaattcatc gatgatatca
     4381 gatccactag tggcctatgc taatgcatat gtgcagtttt cataatctta atagatattg
     4441 tacgtattca cgtgtgaatt gctaaagagg gtagaaatct tcaaaacaag caatttgaaa
     4501 atatttacat aggcaatggt acattctaga aacacgaaaa gccaacaaga taactaattc
     4561 tcaccaaaac catctcctat accaaggctt catatgtgtt gattcattac tgccctttcc
     4621 cctactagta ttgtgggttt tatcctgtac aattaactca tttaattggt gttccaggat
     4681 ttctacctct ttctttagct gactttctct ctcctcatgc tctagtttcc accaagagaa
     4741 gtgaagcagt aaataaacgg aagatcccat tagaatgaac atccaaagag gcctaataaa
     4801 tctttttaca gggatctcat gtcctgattc ttcctcttgc gagaattttt tgagcatatc
     4861 taactccttt ttgatattta attttgtagt atgctcatta atcaaatcgc gtatcagttc
     4921 agtatcaaca ccatccaaag aatccaaatt agccaagtct tctaggctct tgatttcttt
     4981 cttcggtgac gttgaaacag ctcttcgttg ttggttgagg aggcgcctgt ggtaataggc
     5041 aacggttgtt atgggaaaca atttagttct actcaaagta atgagagcat tagagcgtct
     5101 ttttaacata ttgtctatta tatgctttgg ttcccatttc ctttctcaaa gtttacaaat
     5161 ataacagagt gcttagcgaa tactttattt tatgagtctt gccgttagtg accccaccca
     5221 aaataagatg caaaccggta tacatattag acatttccca cgtatacaaa tatattaagt
     5281 aaataagcct cgtcgtgatg aacttaaata catggtattg agagagttat aaacaggtag
     5341 atagatatct taaataaaaa taagcagata agtaacaatg tttaagaaaa cgatggggaa
     5401 gcgctcgagg gggggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg
     5461 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5521 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5581 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5641 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5701 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5761 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5821 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5881 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5941 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6001 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6061 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6121 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6181 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6241 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6301 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6361 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6421 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6481 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6541 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6601 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6661 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6721 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6781 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6841 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6901 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6961 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7021 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7081 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7141 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7201 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7261 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7321 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7381 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7441 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7501 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7561 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7621 gccac
//
