LOCUS       sequence-256856-        7804 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of GFP-PEP12 as an additional copy in genome, use 
            auxotrophic marker URA3(Kluyveromyces lactis).Marker for yeast late 
            endosome..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7804)
  AUTHORS   Zhu J, Zhang ZT, Tang SW, Zhao BS, Li H, Song JZ, Li D, Xie Z
  TITLE     A Validated Set of Fluorescent-Protein-Based Markers for Major 
            Organelles in Yeast (Saccharomyces cerevisiae).
  JOURNAL   MBio. 2019 Sep 3;10(5). pii: mBio.01691-19. doi: 
            10.1128/mBio.01691-19.
  PUBMED    31481383
REFERENCE   2  (bases 1 to 7804)
  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: "MBio."; 
            date: "2019-09-3"; volume: "10(5). pii: mBio.01691-19. doi"; pages: 
            " 10.1128/mBio.01691-19"
COMMENT     Sequence Label: ZJOM157
FEATURES             Location/Qualifiers
     source          1..7804
                     /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"
     promoter        1055..1271
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     protein_bind    complement(2215..2248)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3318..4031
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             3318..4031
                     /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"
     primer_bind     complement(5616..5636)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5616..5634)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5655..5671)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5655..5671)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5668..5690)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5679..5695
                     /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(5703..5709,5710..5727,5728..5733))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5748..5769
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5886..5903)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6057..6645)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6137..6156)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6816..7607,7608..7676))
                     /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     7439..7458
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7677..7781)
                     /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 tgggtacccg
      661 ttgctaaatt cgagtgaaac acaggaagac cagaaaatcc tcatttcatc catattaaca
      721 ataatttcaa atgtttattt gcattatttg aaactaggga agacaagcaa cgaaacgttt
      781 ttgaaaattt tgagtatttt caataaattt gtagaggact cagatattga aaaaaagcta
      841 cagcaattaa tacttgataa gaagagtatt gagaagggca acggttcatc atctcatgga
      901 tctgcacatg aacaaacacc agagtcaaac gacgttgaaa ttgaggctac tgcgccaatt
      961 gaggcctttt ttgcgaggca tatttatggt gaaggataag ttttgaccat caaagaaggt
     1021 taatgtggct gtggtttcag ggtccataaa gcttttcaat tcatcttttt tttttttgtt
     1081 cttttttttg attccggttt ctttgaaatt tttttgattc ggtaatctcc gagcagaagg
     1141 aagaacgaag gaaggagcac agacttagat tggtatatat acgcatatgt ggtgttgaag
     1201 aaacatgaaa ttgcccagta ttcttaaccc aactgcacag aacaaaaacc tgcaggaaac
     1261 gaagataaat cctcgagatg tccacaaaat catataccag tagagctgag actcatgcaa
     1321 gtccggttgc atcgaaactt ttacgtttaa tggatgaaaa gaagaccaat ttgtgtgctt
     1381 ctcttgacgt tcgttcgact gatgagctat tgaaacttgt tgaaacgttg ggtccataca
     1441 tttgcctttt gaaaacacac gttgatatct tggatgattt cagttatgag ggtactgtcg
     1501 ttccattgaa agcattggca gagaaataca agttcttgat atttgaggac agaaaattcg
     1561 ccgatatcgg taacacagtc aaattacaat atacatcggg cgtttaccgt atcgcagaat
     1621 ggtctgatat caccaacgcc cacggggtta ctggtgctgg tattgttgct ggcttgaaac
     1681 aaggtgcgca agaggtcacc aaagaaccaa ggggattatt gatgcttgct gaattgtctt
     1741 ccaagggttc tctagcacac ggtgaatata ctaagggtac cgttgatatt gcaaagagtg
     1801 ataaagattt cgttattggg ttcattgctc agaacgatat gggaggaaga gaagaagggt
     1861 ttgattggct aatcatgacc ccaggtgtag gtttagacga caaaggcgat gcattgggtc
     1921 agcagtacag aaccgtcgac gaagttgtaa gtggtggatc agatatcatc attgttggca
     1981 gaggactttt cgccaagggt agagatccta aggttgaagg tgaaagatac agaaatgctg
     2041 gatgggaagc gtaccaaaag agaatcagcg ctccccatta attatacagg aaacttaata
     2101 gaacaaatca catatttaat ctaatagcca cctgcattgg cacggtgcaa cactcacttc
     2161 aacttcatct tacaaaagat cacgtgatct gttgtattgg gatctctaga cctaataact
     2221 tcgtatagca tacattatac gaagttatat taagggttgt cgacctgcag cgtacatcga
     2281 taagcttgat atcgaattcc tgcagcccgg gggatcctac ttagtccttt ggtcaaaaat
     2341 atttcagaac aaagtgttgt caatgaagga tataattctg atagggtttt gactatgatg
     2401 gacaatggat catttccaac gtctatttct gcctccacaa ttaagctact catgaaatcg
     2461 accactgctt tatcaaagtc tttttggaag cttgattgta attcagcaaa aattataagc
     2521 atcatcgatt ttacttcatc ttcgcttatg cgcaaaataa gttccttcac taaaaatcgc
     2581 acctccttga aattgtactt gaacttattc aatagttgta gaattatgct cagtaaatac
     2641 gtgacgtggg tgtcctcgcc atagtgtatc ctaatttgca gctcatttaa caatgcaatt
     2701 gttgcgtctt tattggaaaa gcaaccaacg aaaacgtgga tggatgatct tgataatatt
     2761 tcaaataaat tgagtgcgaa gggaatatca ttctgtataa gtttagataa atgtacgcgg
     2821 gactcagaat ggtcttggta agacctggtc aatatttctt cgattttctg acgatcttta
     2881 agggcttcac tattcgttaa atcagccggc attttcccag attcagattt ttgccttagc
     2941 tggaaaattg tatttatagc atcattgtat ttttggacat caggggtgga tttcaaagtc
     3001 ttgtcaaatg cagcgcagag gctatcaata gtagagctat cgattggatc attccctttc
     3061 tcacacagtg gcatcgtgat tctaattttt tagtaatctt ttcatgtgag tagtttagat
     3121 aaaacgtctt cactattaag tagaagattc gtaattggat ttaagccttc gtgccttatc
     3181 gcggctgata atcatcatga tcgacgatga actactgcgc cagagtataa tcccctataa
     3241 aacaggcctg ctatcactcc aaagagaatg cttttataga agaatataac gtaaattact
     3301 acaataattg tgttgagatg tctaaaggtg aagaattatt cactggtgtt gtcccaattt
     3361 tggttgaatt agatggtgat gttaatggtc acaaattttc tgtctccggt gaaggtgaag
     3421 gtgatgctac ttacggtaaa ttgaccttaa aatttatttg tactactggt aaattgccag
     3481 ttccatggcc aaccttagtc actactttaa cttatggtgt tcaatgtttt tctagatacc
     3541 cagatcatat gaaacaacat gactttttca agtctgccat gccagaaggt tatgttcaag
     3601 aaagaactat ttttttcaaa gatgacggta actacaagac cagagctgaa gtcaagtttg
     3661 aaggtgatac cttagttaat agaatcgaat taaaaggtat tgattttaaa gaagatggta
     3721 acattttagg tcacaaattg gaatacaact ataactctca caatgtttac atcatggctg
     3781 acaaacaaaa gaatggtatc aaagttaact tcaaaattag acacaacatt gaagatggtt
     3841 ctgttcaatt agctgaccat tatcaacaaa atactccaat tggtgatggt ccagtcttgt
     3901 taccagacaa ccattactta tccactcaat ctgccttatc caaagatcca aacgaaaaga
     3961 gagaccacat ggtcttgtta gaatttgtta ctgctgctgg tattacccat ggtatggatg
     4021 aattgtacaa agcttcgtac gctgcaggtc gacggatccc cgggttaatt aacatgtcgg
     4081 aagacgaatt ttttggtggt gataatgaag ccgtttggaa cggttccaga ttcagtgatt
     4141 cacctgagtt ccaaacgttg aaagaagagg ttgctgcaga gttatttgaa ataaatgggc
     4201 aaataagcac gctgcagcag tttaccgcga cacttaagtc atttatagat cggggagatg
     4261 ttagtgcgaa agttgtggaa agaattaata agagatctgt ggcaaagata gaagaaatag
     4321 gcgggctcat taaaaaggtc aatacatcag tgaaaaagat ggatgcgatt gaggaagcta
     4381 gcctggataa gactcaaata atagcgagag agaaacttgt gagggatgtc agttactctt
     4441 ttcaagagtt tcaaggtatt cagcggcagt ttacccaagt aatgaaacaa gttaatgaaa
     4501 gagcaaaaga atctcttgaa gcaagtgaga tggcaaatga tgctgcttta ttagatgaag
     4561 aacaaaggca gaatagctca aaaagtactc gaataccagg cagccaaata gtcattgaga
     4621 gagacccgat aaataacgaa gagtttgctt atcagcaaaa tcttatcgag caaagagacc
     4681 aggaaatcag caatattgaa agaggtataa cggaactgaa cgaagttttt aaagatttgg
     4741 gaagtgttgt tcaacagcag ggtgtgctag ttgacaatat tgaagcaaat atttatacaa
     4801 cgtcagataa cactcaattg gcttcagacg agctaaggaa ggccatgcgg taccaaaaac
     4861 gtacgagcag atggagggtg tatttgttga ttgtgcttct cgtaatgctt ctttttattt
     4921 ttctcattat gaaattgtaa gcggccgcaa aaaactgtat tataagtaaa tgcatgtata
     4981 ctaaactcac aaattagagc ttcaatttaa ttatatcagt tattacccgg gaatctcggt
     5041 cgtaatgatt tctataatga cgaaaaaaaa aaaattggaa agaaaaagct tcatggcctt
     5101 tataaaaagg aactatccaa tacctcgcca gaaccaagta acagtatttt acggggcaca
     5161 aatcaagaac aataagacag gactgtaaag atggacgcat tgaactccaa agaacaacaa
     5221 gagttccaaa aagtagtgga acaaaagcaa atgaaggatt tcatgcgttt gtactctaat
     5281 ctggtagaaa gatgtttcac agactgtgtc aatgacttca caacatcaaa gctaaccaat
     5341 aaggaacaaa catgcatcat gaagtgctca gaaaagttct tgaagcatag cgaacgtgta
     5401 gggcagcgtt tccaagaaca aaacgctgcc ttgggacaag gcttgggccg ataaggtgta
     5461 ctggcgtata tatatctaat tatgtatctc tggtgtagcc catttttagc atgtaaatat
     5521 aaagagaaac catatctaat ctaaccaaat ccaaacaaaa ttcaatagtt acgtagacgt
     5581 cctacgattc cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
     5641 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5701 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5761 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5821 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5881 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5941 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     6001 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6061 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6121 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6181 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6241 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6301 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6361 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6421 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6481 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6541 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6601 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6661 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6721 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6781 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6841 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6901 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6961 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     7021 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7081 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     7141 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7201 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7261 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7321 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7381 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7441 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7501 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7561 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7621 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7681 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7741 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7801 ccac
//
