LOCUS       sequence-259151-        4711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part of GoldenGate Yarrowia lipolytica toolkit for assembling an 
            expression plasmid for Y. lipolytic yeast. Antibiotic resistance 
            marker for selection after transformation in Y. lipolytica 
            (Marker_Nat).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4711)
  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 4711)
  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: GGE368
FEATURES             Location/Qualifiers
     source          1..4711
                     /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(370..403)
                     /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(1599..1618)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1600..1618)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1625..1642)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1625..1641)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1634..1656)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1779..2081
                     /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     1960..1979
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2430..3224
                     /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             2430..3224
                     /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(2484..2503)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3094..3113
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3424..3433
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3430..3804
                     /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      3942..4530
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4431..4450
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4684..4701
                     /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 gcccttgggg gtctctaggt tagggataac
      361 agggtaatga taacttcgta tagcatacat tatacgaagt tatggggttg gcggcgcatt
      421 tgtgtcccaa aaaacagccc caattgcccc aattgacccc aaattgaccc agtagcggac
      481 ccaaccccgg cgagagcccc cttcacccca catatcaaac ctcccccggt tcccacactt
      541 gccgttaagg gcgtagggta ctgcagtctg gaatctacgc ttgttcagac tttgtactag
      601 tttctttgtc tggccatccg ggtaacccat gccggacgca aaatagacta ctgaaaattt
      661 ttttgctttg tggttgggac tttagccaag ggtataaaag accaccgtcc ccgaattacc
      721 tttcctcttc ttttctctct ctccttgtca actcacaccc gaagcacaat gaccactctg
      781 gatgacaccg cttaccgata ccgaacttcc gttcctggcg atgccgaggc tattgaggct
      841 ctggatggat ctttcaccac tgacaccgtt ttccgagtga ccgctactgg cgacggcttc
      901 accctgcgag aggtgcctgt cgaccctcct ctcaccaagg ttttccctga cgatgagtcg
      961 gacgatgagt ctgacgctgg agaggacggc gaccctgact ctcgaacttt cgtggcttac
     1021 ggcgacgatg gcgacctggc cggctttgtg gtcgtttctt actccggatg gaaccgacga
     1081 ctgaccgtgg aggacatcga ggtcgctcct gagcaccgag gtcatggtgt cggacgagct
     1141 ctgatgggtc tggctactga gttcgctcga gagcgaggtg ctggccacct gtggctcgag
     1201 gtcaccaacg ttaacgcccc tgctattcat gcctaccgac gaatgggttt taccctgtgt
     1261 ggcctcgata ctgccctgta cgacggaacc gcttccgatg gagagcaggc cctctacatg
     1321 tcgatgccct gcccttaagt gtctgtggta tctaagctat ttatcactct ttacaacttc
     1381 tacctcaact atctacttta ataaatgaat atcgtttatt ctctatgatt actgtatatg
     1441 cgttcctcta agacaaatcg aatataactt cgtatagcag gagttatccg aagcgagtag
     1501 ggataacagg gtaatacgga gagaccccca agggcgaatt ctgcagatat ccatcacact
     1561 ggcggccgct cgagcatgca tctagagggc ccaattcgcc ctatagtgag tcgtattaca
     1621 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     1681 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     1741 atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt tacacctata
     1801 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgccgg
     1861 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     1921 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     1981 ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     2041 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc atgagattat
     2101 caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag aaacggtgct
     2161 gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga
     2221 gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga
     2281 cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca
     2341 aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagctctg
     2401 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     2461 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     2521 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     2581 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     2641 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     2701 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     2761 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     2821 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     2881 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     2941 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3001 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3061 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3121 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3181 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt aacgcttaca
     3241 atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatcaggt
     3301 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3361 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata gcacgtgagg
     3421 agggccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc
     3481 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac
     3541 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg
     3601 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag
     3661 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc
     3721 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac
     3781 ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc atttttaatt taaaaggatc
     3841 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3901 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3961 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4021 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4081 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4141 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4201 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4261 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4321 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4381 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4441 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4501 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4561 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4621 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4681 cgcagcgagt cagtgagcga ggaagcggaa g
//
