LOCUS       sequence-128679-        8467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of 2GFP tag at Atg29 C terminus, use auxotrophic marker 
            URA3(Candida albicans).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8467)
  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 8467)
  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: LD229
FEATURES             Location/Qualifiers
     source          1..8467
                     /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             2207..2920
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2207..2920
                     /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             2957..3673
                     /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             2957..3670
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      3694..3881
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3749..3773)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        3956..4299
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      5118..5315
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(6279..6299)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6279..6297)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6318..6334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6318..6334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6331..6353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6342..6358
                     /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(6366..6372,6373..6390,6391..6396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6411..6432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6549..6566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6720..7308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6800..6819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7479..8270,8271..8339))
                     /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     8102..8121
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8340..8444)
                     /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 ccgcggccaa ataagttaaa atctgcggtc cattgtagca agtaaggaat atgtgattca
      721 taaatttcga atttctttcc gaaaatcttg ccatctctaa tcaattccga aatcctactt
      781 agacaagaag gattaagcaa agaaattttg taaaacagat tccagccaac gtggtaacca
      841 taaaatggta taccctttac aacagagaca tcagcgacaa aattgagatt tccaagtttg
      901 tcgccggcta aatcgtgttt atcatctttt ttccttttaa aggatgccct aatttttact
      961 tccagcgttt tatgaacttg ggcacatctt tggtgtctca atgtactcgt gtcagttatt
     1021 tgtccatcat acttgatgaa catgtaaggt agaatgccat ggacgtggca caatacttga
     1081 tgccctgtgg gtaaagcacc aaacaccctg atgtttggga cctgtgaaaa ctggtttaag
     1141 ggcaggcttt caccgtgcga tgggtccaaa aaggtgggtt ttgacatata gtagtcttga
     1201 ttatttagct ggattctaaa atagtctgat ttagaggatg atctaaccgt atcgctctgt
     1261 attgtgtcgt tcgactccct cgacatttcc aaacatttta tttcgccaca acttgtagtt
     1321 ttgtattgac tcaaatactt tctcttgttt tgtgcttcta ttacagagat ggcttttttc
     1381 ttcaagtaca ctatttctag acgcactgca ctcgtcaaaa aaatgattca aggtgaaaat
     1441 aaaggggtgt tagtgaatac ttatcaagaa gagacataca taattgtact gctgtgcatt
     1501 ttcctacttg actttcatga ttatgaatag tacaaacaca gttgtatata tcaaagttaa
     1561 gggtaggaga ccacagggat tcttggatcc tcccaaattt gagtggaatg gaacaaagga
     1621 gcgacagctt tggacaatgg tatcaaattt gaattattca caagatcaaa tagattggca
     1681 gaatttatca aaaatatttg aaacgcctga attttttctt aaaaagagaa catataaatt
     1741 atttgctgaa cacttagagc ttttacaact acaattggag aaaaaaagag atcttgagaa
     1801 gtattcaaat gatcaagtga atgaagggat gtctgattta atacataaat atacccctac
     1861 tttacaaaat gataatctat taaatgtatc cgcaagccca ctgaccacgg aaagacagga
     1921 ttctgaagaa gttgagacag aagtaacaaa tgaggcgtta caacatttgc aaacttccaa
     1981 aattttgaac attcacaaga aaacatcgga tagtgagaat aagcctaatg acaaattgga
     2041 taaggatggt attaataaag aaatggagtg cggtagttca gatgacgatt tatcttcgag
     2101 cttaagtgtt agtaaatctg cgttggaaga agcgctaatg gacagattgc aattcgcttc
     2161 gtacgctgca ggtcgacgga tcggtgacgg tgctggttta attaacatgt ctaaaggtga
     2221 agaattattc actggtgttg tcccaatttt ggttgaatta gatggtgatg ttaatggtca
     2281 caaattttct gtctccggtg aaggtgaagg tgatgctact tacggtaaat tgaccttaaa
     2341 atttatttgt actactggta aattgccagt tccatggcca accttagtca ctactttaac
     2401 ttatggtgtt caatgttttt ctagataccc agatcatatg aaacaacatg actttttcaa
     2461 gtctgccatg ccagaaggtt atgttcaaga aagaactatt tttttcaaag atgacggtaa
     2521 ctacaagacc agagctgaag tcaagtttga aggtgatacc ttagttaata gaatcgaatt
     2581 aaaaggtatt gattttaaag aagatggtaa cattttaggt cacaaattgg aatacaacta
     2641 taactctcac aatgtttaca tcatggctga caaacaaaag aatggtatca aagttaactt
     2701 caaaattaga cacaacattg aagatggttc tgttcaatta gctgaccatt atcaacaaaa
     2761 tactccaatt ggtgatggtc cagtcttgtt accagacaac cattacttat ccactcaatc
     2821 tgccttatcc aaagatccaa acgaaaagag agaccacatg gtcttgttag aatttgttac
     2881 tgctgctggt attacccatg gtatggatga attgtacaaa attccagttc caacgcgtga
     2941 tctagagaag ccattcatgt ctaaaggtga agaattattc actggtgttg tcccaatttt
     3001 ggttgaatta gatggtgatg ttaatggtca caaattttct gtctccggtg aaggtgaagg
     3061 tgatgctact tacggtaaat tgaccttaaa atttatttgt actactggta aattgccagt
     3121 tccatggcca accttagtca ctactttaac ttatggtgtt caatgttttt ctagataccc
     3181 agatcatatg aaacaacatg actttttcaa gtctgccatg ccagaaggtt atgttcaaga
     3241 aagaactatt tttttcaaag atgacggtaa ctacaagacc agagctgaag tcaagtttga
     3301 aggtgatacc ttagttaata gaatcgaatt aaaaggtatt gattttaaag aagatggtaa
     3361 cattttaggt cacaaattgg aatacaacta taactctcac aatgtttaca tcatggctga
     3421 caaacaaaag aatggtatca aagttaactt caaaattaga cacaacattg aagatggttc
     3481 tgttcaatta gctgaccatt atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt
     3541 accagacaac cattacttat ccactcaatc tgccttatcc aaagatccaa acgaaaagag
     3601 agaccacatg gtcttgttag aatttgttac tgctgctggt attacccatg gtatggatga
     3661 attatataaa taaggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt
     3721 attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt
     3781 ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag
     3841 gtatagtatg aggtcgctct tattgaccac acctctaccg gcagatccgc tagggataac
     3901 agggtaatat agatctgttt agcttgcctc gtccccgccg ggtcacccgg ccagcgacat
     3961 ggaggcccag aataccctcc ttgacagtct tgacgtgcgc agctcagggg catgatgtga
     4021 ctgtcgcccg tacatttagc ccatacatcc ccatgtataa tcatttgcat ccatacattt
     4081 tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg ctgcagacct gcgagcaggg
     4141 aaacgctccc ctcacagacg cgttgaattg tccccacgcc gcgcccctgt agagaaatat
     4201 aaaaggttag gatttgccac tgaggttctt ctttcatata cttcctttta aaatcttgct
     4261 aggatacagt tctcacatca catccgaaca taaacaacca tgacagtcaa cactaagacc
     4321 tatagtgaga gagcagaaac tcatgcctca ccagtagcac aacgattatt tcgattaatg
     4381 gaactgaaga aaaccaattt atgtgcatca attgatgttg ataccactaa ggaattcctt
     4441 gaattaattg ataaattggg tccttatgta tgcttaatca agactcatat tgatataatc
     4501 aatgattttt cctatgaatc cactattgaa ccattattag aactttcacg taaacatcaa
     4561 tttatgattt ttgaagatag aaaatttgct gatattggta ataccgtgaa gaaacaatat
     4621 attggtggag tttataaaat tagtagttgg gcagatatta ctaatgctca tggtgtcact
     4681 gggaatggag tagttgaagg attaaaacag ggagctaaag aaaccaccac caaccaagag
     4741 ccaagagggt tattgatgtt agctgaatta tcatcagtgg gatcattagc atatggagaa
     4801 tattctcaaa aaactgttga aattgctaaa tccgataagg aatttgttat tggatttatt
     4861 gcccaacgtg atatgggtgg acaagaagaa ggatttgatt ggcttattat gacacctgga
     4921 gttggattag atgataaagg tgatggatta ggacaacaat atagaactgt tgatgaagtt
     4981 gttagcactg gaactgatat tatcattgtt ggtagaggat tgtttggtaa aggaagagat
     5041 ccagatattg aaggtaaaag gtatagagat gctggttgga atgcttattt gaaaaagact
     5101 ggccaattat aatcagtact gacaataaaa agattcttgt tttcaagaac ttgtcatttg
     5161 tatagttttt ttatattgta gttgttctat tttaatcaaa tgttagcgtg atttatattt
     5221 tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat
     5281 gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc gattcgatac taacgccgcc
     5341 atccagtgtc gaaaacgagc tcgaattcat aacttcgtat aatgtatgct atacgaagtc
     5401 gatgatatca gatccactag tggcctatgc tttacatctt tcacaagcac aatcaaaaaa
     5461 ccaactgtcg gccaaaaagg cccggcgttt tactgttggt aagtccagta ccccggaata
     5521 atcgatacag atctgctcat ccttttttat atctctattc atagtgaata gcattgaatt
     5581 acctttacga tatttcgtta tattggggtt acaagagtgg ttgaaatagg aggcttctgg
     5641 aaatacccag tatccaaaat attctctgct gtctgaggct tcgccctctt gccataaacc
     5701 aaaagcattc ccatattcag tacctaatat atgtctcaat agcggtatgg agagcattct
     5761 atgcaaatgt gaaggtaata gaatatagag tgtttgaaat actaattttt gaaagtgcag
     5821 taagacagga aatttagata ttttgctaag ctcattcgat tgtaacatat tgaaagctcg
     5881 gtatgttatg cactgaggat ccatatattt aagattaaac aaactctcgc acacaaacct
     5941 aatgcagcaa tattcatctt cgcaagtggg tggtaattga tttattctct tcgcagattt
     6001 catgttgttg atgcgcggta tccatttact ttcaatctca tcccatgaag actgtatgac
     6061 attttccgaa attaagatag agtttaattt ttcctcctgt tcagaggtat aattatatct
     6121 tttcagcatg cttggaaaat gatgcagtaa tatctcataa cattcaatta attcaatgat
     6181 atttggtatt tgaagataag aagttctgca atgttctgaa cagaaccaca gtccagcgcc
     6241 cagaactcga gggggggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg
     6301 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     6361 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     6421 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6481 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6541 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6601 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6661 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6721 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6781 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6841 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6901 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6961 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     7021 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     7081 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     7141 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     7201 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     7261 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     7321 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     7381 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     7441 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     7501 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     7561 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7621 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7681 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7741 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7801 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7861 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7921 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7981 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     8041 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     8101 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     8161 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     8221 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     8281 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     8341 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     8401 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     8461 gtgccac
//
