LOCUS       sequence-263168-        4259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part of GoldenGate Yarrowia lipolytica toolkit for assembling an 
            expression plasmid for Y. lipolytic yeast. Coding sequence of 
            fluorescent protein in position 2 (G2_YFP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4259)
  AUTHORS   Larroude M, Park YK, Soudier P, Kubiak M, Nicaud JM, Rossignol T
  TITLE     A modular Golden Gate toolkit for Yarrowia lipolytica synthetic 
            biology.
  JOURNAL   Microb Biotechnol. 2019 May 31. doi: 10.1111/1751-7915.13427.
  PUBMED    31148366
REFERENCE   2  (bases 1 to 4259)
  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: "Microb 
            Biotechnol. 2019 May 31. doi: 10.1111/1751-7915.13427."
COMMENT     Sequence Label: GGE070
FEATURES             Location/Qualifiers
     source          1..4259
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(348..1061,1062..1064,1065..1067))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(393..414)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     742..761
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1001..1022
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1147..1166)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1148..1166)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1173..1190)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1173..1189)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1182..1204)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1327..1629
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1508..1527
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1978..2772
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1978..2772
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2032..2051)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2642..2661
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2972..2981
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2978..3352
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      3490..4078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3979..3998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4232..4249
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gcccttggtc tctatcctta cttgtacagc
      361 tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg
      421 cgcttctcgt tggggtcttt gctcagggcg gactggtagc tcaggtagtg gttgtcgggc
      481 agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg
      541 ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt cttctgcttg
      601 tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg ccccaggatg
      661 ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc
      721 tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc
      781 tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg
      841 gggtagcggg cgaagcactg caggccgtag ccgaaggtgg tcacgagggt gggccagggc
      901 acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg
      961 ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc
     1021 aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccattgt agagaccaag
     1081 ggcgaattct gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc
     1141 aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac
     1201 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     1261 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctatac
     1321 gtacggcagt ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat
     1381 gtacagagtg atattattga cacgccgggg cgacggatgg tgatccccct ggccagtgca
     1441 cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa
     1501 agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa
     1561 gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg
     1621 ggaatataaa tgtcaggcat gagattatca aaaaggatct tcacctagat ccttttcacg
     1681 tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc
     1741 tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg
     1801 cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg
     1861 ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg
     1921 atctgatggc gcaggggatc aagctctgat caagagacag gatgaggatc gtttcgcatg
     1981 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     2041 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     2101 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     2161 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     2221 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     2281 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     2341 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     2401 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     2461 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     2521 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     2581 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     2641 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     2701 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     2761 gagttcttct gaattattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat
     2821 ctgtgcggta tttcacaccg catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2881 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2941 ataaatgctt caataatagc acgtgaggag ggccaccatg gccaagttga ccagtgccgt
     3001 tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg
     3061 gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct
     3121 gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt
     3181 gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga
     3241 cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct
     3301 gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct
     3361 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3421 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3481 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3541 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3601 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     3661 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3721 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3781 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3841 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3901 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3961 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4021 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4081 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4141 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4201 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
