LOCUS       sequence-128691-        6571 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of GFP tag at Atg16 C terminus, use auxotrophic marker 
            URA3(Candida albicans).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6571)
  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 6571)
  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: LD116
FEATURES             Location/Qualifiers
     source          1..6571
                     /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             1259..1969
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1286..1314)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1909..1929
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1993..2180
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2048..2072)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2237..2452
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             2453..3256
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     primer_bind     complement(4383..4403)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4383..4401)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4422..4438)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4422..4438)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4435..4457)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4446..4462
                     /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(4470..4476,4477..4494,4495..4500))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4515..4536
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4653..4670)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4824..5412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4904..4923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5583..6374,6375..6443))
                     /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     6206..6225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6444..6548)
                     /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 ccgcggacac ttgctcgctt ttacttattc tgtttgtagt tgacctttat ttaacgttga
      721 tttttgttat taacctgcgc gaatctgtat taagggcgca tattccatgg gcaatttcat
      781 tataacagaa aggaagaaag caaaggagga aagaagcaat ccacaaacag atagcatgga
      841 tgatttgtta attcgtagac taacagatcg aaacgacaag gaggcccatt taaatgagtt
      901 gtttcaagat aatagtggcg ccattggtgg caacattgtc agccatgacg atgctttatt
      961 gaatacactg gcaattctac aaaaagaact gaaaagtaag gagcaagaaa tacgaagatt
     1021 gaaagaagtt attgcgttga aaaataagaa tacggaaagg ttgaatgacg aattgattag
     1081 tggaaccatt gaaaataatg ttttacaaca aaaactctcg gatctgaaaa aagaacattc
     1141 ccagctagtt gcgagatggc taaaaaagac agagaaagag acagaagcca tgaacagcga
     1201 aatagatgga acgaaagctt cgtacgctgc aggtcgacgg atccccgggt taattaacag
     1261 taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
     1321 taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact
     1381 tacccttaaa tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac
     1441 tactttcact tatggtgttc aatgcttttc aagataccca gatcatatga aacggcatga
     1501 ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga
     1561 tgacgggaac tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag
     1621 aatcgagtta aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga
     1681 atacaactat aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa
     1741 agttaacttc aaaattagac acaacattga agatggaagc gttcaactag cagaccatta
     1801 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
     1861 cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga
     1921 gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaat agggcgcgcc
     1981 acttctaaat aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa
     2041 aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc
     2101 ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagtatga ggtcgctctt
     2161 attgaccaca cctctaccgg cagatccgct agggataaca gggtaatata gatctgagag
     2221 tgcaccatac caccttttca attcatcatt ttttttttat tctttttttt gatttcggtt
     2281 tccttgaaat ttttttgatt cggtaatctc cgaacagaag gaagaacgaa ggaaggagca
     2341 cagacttaga ttggtatata tacgcatatg tagtgttgaa gaaacatgaa attgcccagt
     2401 attcttaacc caactgcaca gaacaaaaac ctgcaggaaa cgaagataaa tcatgtcgaa
     2461 agctacatat aaggaacgtg ctgctactca tcctagtcct gttgctgcca agctatttaa
     2521 tatcatgcac gaaaagcaaa caaacttgtg tgcttcattg gatgttcgta ccaccaagga
     2581 attactggag ttagttgaag cattaggtcc caaaatttgt ttactaaaaa cacatgtgga
     2641 tatcttgact gatttttcca tggagggcac agttaagccg ctaaaggcat tatccgccaa
     2701 gtacaatttt ttactcttcg aagacagaaa atttgctgac attggtaata cagtcaaatt
     2761 gcagtactct gcgggtgtat acagaatagc agaatgggca gacattacga atgcacacgg
     2821 tgtggtgggc ccaggtattg ttagcggttt gaagcaggcg gcagaagaag taacaaagga
     2881 acctagaggc cttttgatgt tagcagaatt gtcatgcaag ggctccctat ctactggaga
     2941 atatactaag ggtactgttg acattgcgaa gagcgacaaa gattttgtta tcggctttat
     3001 tgctcaaaga gacatgggtg gaagagatga aggttacgat tggttgatta tgacacccgg
     3061 tgtgggttta gatgacaagg gagacgcatt gggtcaacag tatagaaccg tggatgatgt
     3121 ggtctctaca ggatctgaca ttattattgt tggaagagga ctatttgcaa agggaaggga
     3181 tgctaaggta gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagaagatg
     3241 cggccagcaa aactaaaaaa ctgtattata agtaaatgca tgtatactaa actcacaaat
     3301 tagagcttca atttaattat atcagttatt accctatgcg gtgtgaaata ccgcacagat
     3361 gcgtaaggag aaaataccgc atcaggaaat tgtaaacgtt aatattttgt taaaattcgc
     3421 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3481 ttataaatca aaagaataga ccgagatagg gttgagatcg atgatatcag atccactagt
     3541 ggcctatgct gatgcatcag ttgtcaaatc gtcacaaaat gcatacaaaa gaaaataaaa
     3601 tcattgtggt gcatatacat ggacagaata tgaaacgcca aagtgtttgc aataaagagg
     3661 tgaaaatatg ttaaacatgc tatagagagt acacctgcaa gttaagaaaa tgccattcgt
     3721 gtttttatct actataatgt aatgtgacag ctgttttaaa cctttcttaa ggtcgtttgt
     3781 gcgagaacag gctttttttt tggagaatgt atcttgagag aaactcaata gtaataactg
     3841 gatgcataaa gaaaatttca tatgaatcac tacaaatctt tggcgttaga attctattgg
     3901 gtaaattgaa ctttgtagct ttttgatggt aaacttggcg gttttgcgcg acccatgatc
     3961 ctaagaaacc cgcagtttct attaggattc acgatggtct ttatctcacc ttctattatg
     4021 tgtaagttga aatgcccctt gctgttctta tagcagaaag cttttttttt catttatagg
     4081 actagcattt tggcattgga agggaaaaaa tcatgctttc attgaaaaat gaaaagaaac
     4141 atatcgttat tttgtgggaa cactgaagta taattttttg tttcggaaat ttaccattgc
     4201 atagtgggta ttttcatcga aagttggcta aagaatgcat gcggcctgtt cattctggtt
     4261 atgagcattt caattggtta gtatattggc gtgagtggaa agaatagtgg ataaaaacta
     4321 agagacccgt tcattctcct gatacgatcc tcgagggggg gcccggtacc cagcttttgt
     4381 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     4441 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4501 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4561 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4621 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4681 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4741 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4801 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4861 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4921 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4981 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5041 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5101 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5161 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5221 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5281 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5341 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5401 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5461 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5521 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5581 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5641 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5701 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5761 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5821 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5881 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5941 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6001 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6061 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6121 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6181 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6241 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6301 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6361 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6421 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6481 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6541 gttccgcgca catttccccg aaaagtgcca c
//
