LOCUS       sequence-263228-        4587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part of GoldenGate Yarrowia lipolytica toolkit for assembling an 
            expression plasmid for Y. lipolytic yeast. Sequence for homologous 
            integration into Y. lipolytica genome. Linearization of cassette by 
            NotI restriction enzyme after the assembly.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4587)
  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 4587)
  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: GGE209
FEATURES             Location/Qualifiers
     source          1..4587
                     /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(1475..1494)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1476..1494)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1501..1518)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1501..1517)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1510..1532)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1655..1957
                     /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     1836..1855
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2306..3100
                     /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             2306..3100
                     /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(2360..2379)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2970..2989
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3300..3309
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3306..3680
                     /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      3818..4406
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4307..4326
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4560..4577
                     /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 gcccttcact gagcctccac ctagcctggg
      361 gtctctacct tgtgtgtgtg tgttgaataa atagattgtg ttaataacgt atcttctacc
      421 tcaattgagt tagcaggtcc ggtataactt gatctagaaa gcagcaagta tggaggggtg
      481 gggcaggtta tatactgggg tcatgcagtg gtcactttgg cttcctatgg ggtttatggt
      541 ttcgttgtcc cgatttagac tctgcctcgg ctcaagcaag tcgctcgtca tcgggcgggc
      601 gtaatccatg ttggcaatac aaccgcaacc acaatgaatt tagggagaaa agggtcctca
      661 attgttcaga gctatcttgt gtgattccat tgtgaatttt tccgtatcaa tatggagtac
      721 aaatattcgg ttgttctcat tttctcgaaa atgtcatcaa tttcaaagtt gtagaacccc
      781 acatccatac agaagcttgt ggaaatttca acatgaacca ggaaatggtt ataaccgatt
      841 ttacgtttaa ataattctga ctgggaaacg gcatgaccta cgaaatcctt ctcctcttct
      901 tggagggttt ccccatctct ccccaaagag gttggctcgg ggttggccaa cctccccatg
      961 gggatctgcc cgttttcatg ctcactctgg cttgcattag ctcatccaat ccaaataagc
     1021 cccgtgctgg aactcaaccc cacattacac aaatcagccg ttggcctgcg acaacggaaa
     1081 tgattactag acaattgctg ccacttgaca catgtggtaa tagtgacaga aatgacaatt
     1141 gcgacagagt agcccatcaa ttgtaggtct atctatgttc cactagggaa cgccacccat
     1201 cgtaactccg aaattcagat tttttacctt ttctcggtaa ttctcatatt tataccgaaa
     1261 acactggtca ttgcagtact ccctacagtc caataggcct acttctacca gtactgtagt
     1321 cttactctaa ttcgctgctg gagtcaacat ctctttggct tgtacgtcgt aggcagagac
     1381 cccaggtcag gcggaatggc acttcaaggg cgaattctgc agatatccat cacactggcg
     1441 gccgctcgag catgcatcta gagggcccaa ttcgccctat agtgagtcgt attacaattc
     1501 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     1561 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     1621 cccttcccaa cagttgcgca gcctatacgt acggcagttt aaggtttaca cctataaaag
     1681 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgccggggcg
     1741 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     1801 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     1861 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     1921 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggcatga gattatcaaa
     1981 aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac ggtgctgacc
     2041 ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa
     2101 gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc
     2161 aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt
     2221 aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca
     2281 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     2341 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     2401 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     2461 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     2521 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     2581 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     2641 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     2701 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     2761 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     2821 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     2881 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     2941 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3001 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     3061 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt
     3121 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca
     3181 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3241 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac gtgaggaggg
     3301 ccaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     3361 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     3421 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     3481 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     3541 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     3601 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     3661 tggccgagga gcaggactga cacgtgctaa aacttcattt ttaatttaaa aggatctagg
     3721 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3781 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3841 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3901 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3961 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4021 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4081 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4141 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4201 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4261 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4321 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4381 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4441 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4501 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4561 gcgagtcagt gagcgaggaa gcggaag
//
