LOCUS       sequence-263208-        4553 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 4553)
  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 4553)
  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: GGE252
FEATURES             Location/Qualifiers
     source          1..4553
                     /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(1441..1460)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1442..1460)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1467..1484)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1467..1483)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1476..1498)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1621..1923
                     /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     1802..1821
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2272..3066
                     /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             2272..3066
                     /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(2326..2345)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2936..2955
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3266..3275
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3272..3646
                     /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      3784..4372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4273..4292
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4526..4543
                     /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 gtctctgagt gcagaagttg
      361 agtgtttgaa tgtactactt gatagtaggg agaaggaaaa acctggtaat taatatattg
      421 tcatgataac gatactcaag aagaggacga gtgatgcagc agtggaacga gtagttcgat
      481 gtgaggcaaa cgtagcctgc tagttacgta ccggtattgg aaccaatacg aaagtgaaaa
      541 cgcacacatg ttcagatgtt cttgcatcat gaagtgtcaa tattatctca ccatgtcttg
      601 aaatagtaca gtaagcaggt ttagatactg ttccatgttg cattaataca tcgtcgagat
      661 acaattatga tatagagcta cagtatggct ctgaaaagac atgtatcgaa agtaggatag
      721 agtctaagca caagtaacga gctctgcgtt cgattcatgt ctcaaattgt atacgcatag
      781 ccatctatga cttgcgtcac cgccgtcggt tgtctacttt cttgtctcat tcattgatga
      841 tataaataac ctaatcaaca tgctatgttc tcgcaacccg ctcctccttc catctttgtg
      901 ctctctcgtg tcgcagatgg tatcgtgagc gagcctgtct gtagtcggcc acaaattggg
      961 ccacgtcggt gctatcggat cctagaaacg actctcgaag aatctcacta agctcgtccg
     1021 attccacagc tgaatgggtc atactcgact gtagagctgg acttgaaaac ggttgcagag
     1081 cccggtacat ttctgcctcc agctggaccc attctccttc caatttcttg cttgcagtca
     1141 ccttttctcc tactcgttga cgcagttcag gaagagtctc ctggtactgt tccttgagtt
     1201 tttgtgctcg ctgttcgaga tctttgagcg cgcgtgtctg tcggctgtag tctccgccgt
     1261 tctcggccac aattgagccc ttgagcacgt cctgaggcac actggaaatg tgctgggtgg
     1321 ccagctcatc gagatgcgac agtgtgtacg gcggccgctg cgagagaccg gaagggcgaa
     1381 ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg gcccaattcg
     1441 ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa
     1501 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     1561 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct atacgtacgg
     1621 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag
     1681 agtgatatta ttgacacgcc ggggcgacgg atggtgatcc ccctggccag tgcacgtctg
     1741 ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg
     1801 cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct
     1861 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata
     1921 taaatgtcag gcatgagatt atcaaaaagg atcttcacct agatcctttt cacgtagaaa
     1981 gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca
     2041 agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag
     2101 ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct
     2161 ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga
     2221 tggcgcaggg gatcaagctc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     2281 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     2341 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2401 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     2461 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     2521 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     2581 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     2641 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     2701 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     2761 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     2821 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     2881 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     2941 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3001 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3061 ttctgaatta ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc
     3121 ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3181 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3241 gcttcaataa tagcacgtga ggagggccac catggccaag ttgaccagtg ccgttccggt
     3301 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
     3361 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
     3421 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
     3481 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
     3541 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
     3601 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctaaaact
     3661 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3721 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3781 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3841 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3901 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     3961 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4021 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4081 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4141 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4201 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4261 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4321 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4381 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4441 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4501 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aag
//
