LOCUS       sequence-259319-        3936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part of GoldenGate Yarrowia lipolytica toolkit for assembling an 
            expression plasmid for Y. lipolytic yeast. Promoter for gene 
            expression in position 2 (P2_Tef).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3936)
  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 3936)
  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: GGE006
FEATURES             Location/Qualifiers
     source          1..3936
                     /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"
     primer_bind     complement(824..843)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(825..843)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(850..867)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(850..866)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(859..881)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1004..1306
                     /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     1185..1204
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1655..2449
                     /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             1655..2449
                     /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(1709..1728)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2319..2338
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2649..2658
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2655..3029
                     /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      3167..3755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3656..3675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3909..3926
                     /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 tctgcttgac gggttggcgg
      361 cgtatttgtg tcccaaaaaa cagccccaat tgccccaatt gaccccaaat tgacccagta
      421 gcgggcccaa ccccggcgag agcccccttc accccacata tcaaacctcc cccggttccc
      481 acacttgccg ttaagggcgt agggtactgc agtctggaat ctacgcttgt tcagactttg
      541 tactagtttc tttgtctggc catccgggta acccatgccg gacgcaaaat agactactga
      601 aaattttttt gctttgtggt tgggacttta gccaagggta taaaagacca ccgtccccga
      661 attacctttc ctcttctttt ctctctctcc ttgtcaactc acacccgaaa tcgttaagca
      721 tttccttctg agtataagaa tcaacaaaga gaccaagggc gaattctgca gatatccatc
      781 acactggcgg ccgctcgagc atgcatctag agggcccaat tcgccctata gtgagtcgta
      841 ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
      901 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
      961 caccgatcgc ccttcccaac agttgcgcag cctatacgta cggcagttta aggtttacac
     1021 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
     1081 gccggggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
     1141 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
     1201 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
     1261 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggcatgag
     1321 attatcaaaa aggatcttca cctagatcct tttcacgtag aaagccagtc cgcagaaacg
     1381 gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc
     1441 aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt
     1501 atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc
     1561 ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag
     1621 ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     1681 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     1741 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     1801 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     1861 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     1921 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     1981 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     2041 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     2101 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     2161 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     2221 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     2281 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     2341 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     2401 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc
     2461 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     2521 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2581 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg
     2641 tgaggagggc caccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
     2701 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg
     2761 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
     2821 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
     2881 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
     2941 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
     3001 tgcacttcgt ggccgaggag caggactgac acgtgctaaa acttcatttt taatttaaaa
     3061 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3121 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3181 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3241 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3301 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3361 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3421 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3481 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3541 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3601 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3661 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3721 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3781 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3841 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3901 ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
