LOCUS       sequence-316750-        3991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part of GoldenGate Yarrowia lipolytica toolkit for assembling an 
            expression plasmid for Y. lipolytic yeast. Inducible promoter for 
            gene expression in position 2 (P2_EYK300-4AB).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3991)
  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 3991)
  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: GGE139
FEATURES             Location/Qualifiers
     source          1..3991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(416..435)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(417..435)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(442..459)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(442..458)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(451..473)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             596..898
                     /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     777..796
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1247..2041
                     /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             1247..2041
                     /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(1301..1320)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1911..1930
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2241..2250
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2247..2621
                     /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      2759..3347
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3248..3267
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3501..3518
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3635..3656
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3671..3676,3677..3694,3695..3701)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3709..3725
                     /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     3714..3736
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3733..3749
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3733..3749
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3767..3785
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3767..3784
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gttttcagct ttgcaaaagt ttgggaccgc ggtacgtggt ggccttgcct cgaaataata
       61 gtctttgtcc gcactagtca ggtcatcgga ttatgcaagg ctgtggagct taatgcggcg
      121 gctttccctt ttgggattcc gcttcccgct gtggagctta atgcggcggc tttccctttt
      181 gggattccgc ttcccgctgt ggagcttaat gcggcggctt tcccttttgg gattccgctt
      241 cccgctgtgg agcttaatgc ggcggctttc ccttttggga ttccgcttcc ccagattgaa
      301 acgtacagta tagagaaaag tagatgcatc gataagcaga gaccccaagg gcgaattctg
      361 cagatatcca tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta
      421 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
      481 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
      541 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt
      601 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga
      661 tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc
      721 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat
      781 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct
      841 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat
      901 gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag
      961 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga
     1021 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga
     1081 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa
     1141 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg
     1201 caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     1261 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     1321 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     1381 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     1441 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     1501 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     1561 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     1621 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     1681 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     1741 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     1801 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     1861 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     1921 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     1981 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     2041 aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2101 ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2161 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2221 aataatagca cgtgaggagg gccaccatgg ccaagttgac cagtgccgtt ccggtgctca
     2281 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
     2341 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
     2401 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
     2461 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
     2521 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
     2581 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta aaacttcatt
     2641 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2701 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2761 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2821 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2881 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     2941 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3001 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3061 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3121 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3181 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3241 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3301 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3361 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3421 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3481 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     3541 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     3601 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     3661 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     3721 aacaatttca cacaggaaac agctatgacc atgattacgc caagctattt aggtgacact
     3781 atagaatact caagctatgc atcaagcttg gtaccgagct cggatccact agtaacggcc
     3841 gccagtgtgc tggaattcgc ccttccggtc tctttgtgga tccagtagat gtgtaagtgt
     3901 gtagaagtgt cgtgtgggcc aattgtgggg aaaggagctt cttatagaag ctgatctgcc
     3961 cctctttctg atccacgttg acccccacgc t
//
