LOCUS       sequence-259143-        5842 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_Lys5).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5842)
  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 5842)
  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: GGE176
FEATURES             Location/Qualifiers
     source          1..5842
                     /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    2597..2630
                     /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(2730..2749)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2731..2749)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2756..2773)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2756..2772)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2765..2787)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2910..3212
                     /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     3091..3110
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3561..4355
                     /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             3561..4355
                     /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(3615..3634)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4225..4244
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4555..4564
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4561..4935
                     /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      5073..5661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5562..5581
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5815..5832
                     /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 tctccgttag gatatcccgg
      361 atccgtggga atcgatctag ggataacagg gtaattatcg cttcggataa ctcctgctat
      421 acgaagttat acgaattccg gcaaataagc agacttgaag acacctggtg ggtgtgccat
      481 taccattacc ataccgtcgc tacataggtt ttattcatcc actgcttcca atagtgtata
      541 ttacatgctg ttcaattata tgtatctatc tacattgcag aggcatgaac cgtgctccta
      601 cagtacggat gatgttccac tcttcagagc ggactcagtt gatgcagacc gtgtgttgtg
      661 aggacttggc ctccagctgt gtacgacgtc gcgcaagtct ggctgagtat gtgtgtgtgt
      721 gtgtgtgtgt gtgtgtgtgt gtgtgtgcac gtgtgtttat gtgctgccga taggttcaaa
      781 ttctagacgt gcaaattaga ccgggttgga atgaggctgc atggccacag ccggtccaaa
      841 aggcaacgct ctgctcatca actcctccac cgttaaattt ctcagtcgca cctgctccac
      901 ctcattcaca ccctccttgt gtgtcgccag agcgatcaca tgattgttgt tgagcatgaa
      961 aatctcgtgg tgccatctgt cctgtgcgac tccatcaatc cacagctgcg cagagttgct
     1021 ccagcctctc tggacagcca cggcgcccac aacgtcggtt ccggtcttgt ccttggccgc
     1081 gtccatcagt ctggccgtgt ccaggtttct gaactctagc ttggcaaggt ccatgttgag
     1141 ccccatgcca atggccttgg aatacgactc cttgagtgcc caccgcaaga agaactcgcc
     1201 attgccggtg ctgtcgctgg tgacctgaga gtactcgaac ggcgagaaaa tctgtgaaaa
     1261 gccctccatc cagttcatag tccagtttga gggaggaaca gtcgaggtga tatccactcc
     1321 aatctgcttg ccatcaggac gaacagccac aaccaccagc tcgctgtggt gactgatgtt
     1381 gtagtcgaac ggcgagttgg ccacaaatga cttgccctgc ggagtcttcg aaatgacaat
     1441 gtccgtccac ttcttgcctg tcagaacggt ggcaatatac cgctggagca gctgagagcc
     1501 gagagccatc ttcgcatcgc ttctgtaccg aaaacgcgaa atgtcgagct gagattcgac
     1561 aggaagcagt cgcatggcct ggttgaagtc gttctgggag ttccagatgg gccgcacgtc
     1621 cagcagaaac aaatagacgg attgcttgga gttttgccaa tcaaaaagct gtgttagtat
     1681 tgaaggctga aggtgtgtgg acgtacatca ggaacaaaac tccgaggaga gcaggagaaa
     1741 cgcagagcac aacccccatc gccacagaat attggccatg tcgtcgctac aagtctggca
     1801 gtcaccatcg ttatgtgaaa agtgtacttt ggcacacgag gtgaacattt atgtccttga
     1861 tgaaaacgaa aaatgcaaaa tcaaaaccaa aaaaacacca accacgcatg caaacaacaa
     1921 cgatggtaag acagatgttg gcagttggag agaaaaacaa aaaaaaaaac agacaaagct
     1981 cggtgcgtgt gaaagacaca ctgtggaagt gggggcgagg ggcggattga aattgtaagg
     2041 ccaaccctgg ggagagatat cacaaaaaat atcacacgca cgcgttgggg tgggggggta
     2101 aacttcagct gtaagacaga aaaaccctga gtcatctgcc catacccatt tacaccttca
     2161 tggcgcaatt gggtggcaag aaccgttcca acacccagag caatgcgtgt gtgtatgtga
     2221 gtctggcaat gaggcggtcg caaaaacaag ccaaggcact aaaaaaaaca cccccttcaa
     2281 gtccacttgc acttacatgt gcaacttccg cacttccacc tgcactgtac tagacaaccg
     2341 cactactaat cacgctacta ggccgccacc ataaaaaaat cacttggtta gcggaagcag
     2401 tcccagttaa ttagtagtag cagcataagt agctgagcca gtagttgcag aagtggctcc
     2461 ttatggctgt gcatcggtcg attcacgtac acttgctcgt atcatcaatg cacgttcctg
     2521 taataggccg ctcgtatcag acaggcgtcc gtggcgatcc ttgtgtcaat actcacattt
     2581 ccacccggaa ttcagaataa cttcgtatga tgtatgctat acgaagttat gtagggataa
     2641 cagggtaata cctagagacc aagggcgaat tctgcagata tccatcacac tggcggccgc
     2701 tcgagcatgc atctagaggg cccaattcgc cctatagtga gtcgtattac aattcactgg
     2761 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     2821 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     2881 cccaacagtt gcgcagccta tacgtacggc agtttaaggt ttacacctat aaaagagaga
     2941 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccg gggcgacgga
     3001 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc
     3061 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
     3121 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
     3181 acgccattaa cctgatgttc tggggaatat aaatgtcagg catgagatta tcaaaaagga
     3241 tcttcaccta gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga
     3301 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg
     3361 tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg
     3421 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact
     3481 ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga
     3541 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3601 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3661 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3721 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     3781 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3841 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3901 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3961 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4021 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4081 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4141 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4201 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4261 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4321 tcgccttcta tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga
     4381 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatcagg tggcactttt
     4441 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4501 ccgctcatga gacaataacc ctgataaatg cttcaataat agcacgtgag gagggccacc
     4561 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
     4621 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
     4681 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
     4741 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
     4801 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
     4861 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
     4921 gaggagcagg actgacacgt gctaaaactt catttttaat ttaaaaggat ctaggtgaag
     4981 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5041 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5101 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5161 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5221 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5281 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5341 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5401 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5461 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5521 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5581 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5641 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5701 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5761 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5821 tcagtgagcg aggaagcgga ag
//
