LOCUS       sequence-259146-        5458 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_Leu2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5458)
  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 5458)
  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: GGE105
FEATURES             Location/Qualifiers
     source          1..5458
                     /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(387..420)
                     /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(2346..2365)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2347..2365)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2372..2389)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2372..2388)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2381..2403)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2526..2828
                     /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     2707..2726
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3177..3971
                     /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             3177..3971
                     /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(3231..3250)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3841..3860
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4171..4180
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4177..4551
                     /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      4689..5277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5178..5197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5431..5448
                     /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 gccctttgga attcgccctt gcggccggtc
      361 tctaggtatt accctgttat ccctacataa cttcgtatag catacattat acgaagttat
      421 tctgaattcc gcctgagtca tcatttattt accagttggc cacaaaccct tgacgatctc
      481 gtatgtcccc tccgacatac tcccggccgg ctgggtacgt tcgatagcgc tatcggcatc
      541 gacaaggttt gggtccctag ccgataccgc actacctgag tcacaatctt cggaggttta
      601 gtcttccaca tagcacgggc aaaagtgcgt atatatacaa gagcgtttgc cagccacaga
      661 ttttcactcc acacaccaca tcacacatac aaccacacac atccacaatg gaacccgaaa
      721 ctaagaagac caagactgac tccaagaaga ttgttcttct cggcggcgac ttctgtggcc
      781 ccgaggtgat tgccgaggcc gtcaaggtgc tcaagtctgt tgctgaggcc tccggcaccg
      841 agtttgtgtt cgaggaccga ctcattggag gagctgccat tgagaaggag ggcgagccca
      901 tcaccgacgc tactctcgac atctgccgaa aggctgactc tattatgctc ggtgctgtcg
      961 gaggcgctgc caacaccgta tggaccactc ccgacggacg aaccgacgtg cgacccgagc
     1021 agggcctcct caagctgcga aaggacctga acctgtacgc caacctgcga ccctgccagc
     1081 tgctgtcgcc caagctcgcc gatctctccc ccatccgaaa cgttgagggc accgacttca
     1141 tcattgtccg agagctcgtc ggaggtatct actttggaga gcgaaaggag gatgacggat
     1201 ctggcgtcgc ttccgacacc gagacttact ccgttcctga ggttgagcga attgcccgaa
     1261 tggccgcctt cctggccctt cagcacaacc cccctcttcc cgtgtggtcc cttgacaagg
     1321 ccaacgtgct ggcctcctct cgactttggc gaaagactgt cacccgagtc ctcaaggacg
     1381 agttccccca gctggagctc aaccaccagc tgatcgactc ggccgccatg atcctcatca
     1441 agcagccctc caagatgaat ggtatcatca tcaccaccaa catgtttggc gatatcatct
     1501 ccgacgaggc ctccgtcatc cccggttctc tgggtctgct gccctccgcc tctctggctt
     1561 ctctgcccga caccaacgag gcgttcggtc tgtacgagcc ctgtcacgga tctgcccccg
     1621 atctcggcaa gcagaaggtc aaccccattg ccaccattct gtctgccgcc atgatgctca
     1681 agttctctct taacatgaag cccgccggtg acgctgttga ggctgccgtc aaggagtccg
     1741 tcgaggctgg tatcactacc gccgatatcg gaggctcttc ctccacctcc gaggtcggag
     1801 actttgttgc caacaaggtc aaggagctgc tcaagaagga gtaagtcgtt tctacgacgc
     1861 attgatggaa ggagcaaact gacgcgcctg cgggttggtc taccggcagc agccctgcta
     1921 atgaaatgat gatttataat ttaccggtgt agcaaccttg actagaagaa gcagattggg
     1981 tgtgtttgta gtggaggaca gtggtacgtt ttggaaacag tcttcttgaa agtgtcttgt
     2041 ctacagtata ttcactcata acctcaatag ccaagggtgt agtcggttta ttaaaggaag
     2101 ggagttgtgg ctgatgtgga tagatatctt taaagctggc gactgcaccc aacgagtgtg
     2161 gtggtagctt gttactgtat attcgaattc gtataacttc gtatagcagg agttatccga
     2221 agcgataatt accctgttat ccctaacgga gagaccggcc gctcgagcat gcatctaagg
     2281 gcgaattctg cagatatcca tcacactggc ggccgctcga gcatgcatct agagggccca
     2341 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
     2401 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     2461 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg
     2521 tacggcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg
     2581 tacagagtga tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac
     2641 gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa
     2701 gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag
     2761 tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg
     2821 gaatataaat gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt
     2881 agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct
     2941 ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc
     3001 gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc
     3061 cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga
     3121 tctgatggcg caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga
     3181 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3241 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3301 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     3361 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3421 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3481 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3541 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3601 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3661 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     3721 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3781 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3841 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3901 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3961 agttcttctg aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
     4021 tgtgcggtat ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4081 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4141 taaatgcttc aataatagca cgtgaggagg gccaccatgg ccaagttgac cagtgccgtt
     4201 ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg
     4261 ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg
     4321 ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg
     4381 cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac
     4441 gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg
     4501 cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta
     4561 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4621 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4681 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4741 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4801 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4861 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4921 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4981 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5041 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5101 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5161 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5221 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5281 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5341 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5401 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
