LOCUS       sequence-259137-        4836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part of GoldenGate Yarrowia lipolytica toolkit for assembling an 
            expression plasmid for Y. lipolytic yeast. Auxotrophy marker for 
            selection after transformation in Y. lipolytica (Marker_URA3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4836)
  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 4836)
  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: GGE083
FEATURES             Location/Qualifiers
     source          1..4836
                     /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"
     protein_bind    complement(348..381)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1724..1743)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1725..1743)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1750..1767)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1750..1766)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1759..1781)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1904..2206
                     /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     2085..2104
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2555..3349
                     /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             2555..3349
                     /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(2609..2628)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3219..3238
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3549..3558
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3555..3929
                     /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      4067..4655
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4556..4575
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4809..4826
                     /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 tctaggtata acttcgtata
      361 gcatacatta tacgaagtta ttctgaattc cgagaaacac aacaacatgc cccattggac
      421 agatcatgcg gatacacagg ttgtgcagta tcatacatac tcgatcagac aggtcgtctg
      481 accatcatac aagctgaaca agcgctccat acttgcacgc tctctatata cacagttaaa
      541 ttacatatcc atagtctaac ctctaacagt taatcttctg gtaagcctcc cagccagcct
      601 tctggtatcg cttggcctcc tcgataggat ctcggttctg gccgtacaga cctcggccga
      661 caattatgat atccgttccg gtagacatga catcctcaac agttcggtac tgctgtccga
      721 gagcgtctcc cttgtcgtca agacccaccc cgggggtcag aataagccag tcctcagagt
      781 cgcccttagg tcggttctgg gcaacgaagc caaccacaaa ctcggggtcg gatcgggcaa
      841 gctcaatggt ctgcttggag tactcgccag tggccagaga gcccttgcaa gacagctcgg
      901 ccagcatgag cagacctctg gccagcttct cgttgggaga ggggaccagg aactccttgt
      961 actgggagtt ctcgtagtca gagacgtcct ccttcttctg ttcagagaca gtttcctcgg
     1021 caccagctcg caggccagca atgattccgg ttccgggtac accgtgggcg ttggtgatat
     1081 cggaccactc ggcgattcgg tagacaccgt tcttgtactg gtgcttgaca gtgttgccaa
     1141 tatctgcgaa ctttctgtcc tcgaacagga agaaaccgtg cttaagagca agttccttga
     1201 gggggagcac agtgctggcg taggtgaagt cgtcaatgat gtcgatatgg gtcttgatca
     1261 tgcacacata aggtccgacc ttatcggcaa gctcaatgag ctccttggtg gtggtaacat
     1321 ccagagaagc acacaggttg gttttcttgg ctgccacgag cttgagcact cgagcggcaa
     1381 aggcggactt gtggacgtta gctcgagctt cgtaggaggg cattttggtg gtgaagagga
     1441 gactgaaata aatttagtct gcagaacttt ttatcggaac cttatctggg gcagtgaagt
     1501 atatgttatg gtaatagtta cgagttagtt gaacttatag atagactgga ctatacggct
     1561 atcggtccaa attagaaaga acgtcaatgg ctctctgggc gcgaattcgt ataacttcgt
     1621 atagcaggag ttatccgaag cgaacggaga gaccaagggc gaattctgca gatatccatc
     1681 acactggcgg ccgctcgagc atgcatctag agggcccaat tcgccctata gtgagtcgta
     1741 ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     1801 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     1861 caccgatcgc ccttcccaac agttgcgcag cctatacgta cggcagttta aggtttacac
     1921 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
     1981 gccggggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
     2041 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
     2101 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
     2161 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggcatgag
     2221 attatcaaaa aggatcttca cctagatcct tttcacgtag aaagccagtc cgcagaaacg
     2281 gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc
     2341 aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt
     2401 atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc
     2461 ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag
     2521 ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     2581 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     2641 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     2701 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     2761 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     2821 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     2881 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     2941 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3001 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3061 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3121 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     3181 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     3241 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     3301 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc
     3361 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3421 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3481 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg
     3541 tgaggagggc caccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
     3601 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg
     3661 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
     3721 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
     3781 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
     3841 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
     3901 tgcacttcgt ggccgaggag caggactgac acgtgctaaa acttcatttt taatttaaaa
     3961 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4021 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4081 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4141 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4201 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4261 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4321 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4381 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4441 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4501 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4561 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4621 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4681 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4741 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4801 ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
