LOCUS       sequence-259150-        5166 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_hph).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5166)
  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 5166)
  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: GGE367
FEATURES             Location/Qualifiers
     source          1..5166
                     /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)."
     CDS             768..1793
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(2054..2073)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2055..2073)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2080..2097)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2080..2096)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2089..2111)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2234..2536
                     /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     2415..2434
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2885..3679
                     /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             2885..3679
                     /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(2939..2958)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3549..3568
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3879..3888
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3885..4259
                     /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      4397..4985
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4886..4905
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5139..5156
                     /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 tatgggttgg cggcgcattt
      421 gtgtcccaaa aaacagcccc aattgcccca attgacccca aattgaccca gtagcggacc
      481 caaccccggc gagagccccc ttcaccccac atatcaaacc tcccccggtt cccacacttg
      541 ccgttaaggg cgtagggtac tgcagtctgg aatctacgct tgttcagact ttgtactagt
      601 ttctttgtct ggccatccgg gtaacccatg ccggacgcaa aatagactac tgaaaatttt
      661 tttgctttgt ggttgggact ttagccaagg gtataaaaga ccaccgtccc cgaattacct
      721 ttcctcttct tttctctctc tccttgtcaa ctcacacccg aagcacaatg aagaagcccg
      781 agctgaccgc tacctctgtt gagaagttcc tgattgagaa gtttgattcc gtttccgacc
      841 tgatgcagct gtccgagggc gaggagtctc gagccttctc ctttgacgtg ggcggacgag
      901 gttacgttct gcgagtgaac tcgtgtgccg acggcttcta caaggatcga tacgtctacc
      961 gacactttgc ttctgccgct ctgcccatcc ctgaggttct cgacattggc gagttctctg
     1021 agtccctcac ctactgcatc tctcgacgag ctcagggagt caccctgcag gacctccctg
     1081 agactgagct gcctgctgtc ctccagcctg ttgctgaggc catggacgct atcgctgctg
     1141 ctgatctgtc ccagacctcg ggtttcggcc cctttggacc tcagggaatt ggacagtaca
     1201 ccacttggcg agacttcatc tgtgctattg ccgatcctca cgtctaccat tggcagaccg
     1261 ttatggacga tactgtgtcg gcttctgtcg ctcaggctct ggacgagctg atgctctggg
     1321 ccgaggattg ccccgaggtt cgacacctgg tgcatgctga cttcggttcc aacaacgttc
     1381 tcaccgacaa cggccgaatc actgccgtga ttgactggtc cgaggctatg tttggcgact
     1441 cgcagtacga ggtggccaac atcttctttt ggcgaccctg gctggcttgt atggagcagc
     1501 agacccgata cttcgagcga cgacatcctg agctcgctgg ctcccctcga ctgcgagctt
     1561 acatgctccg aattggtctg gaccagctct accagtcgct ggtggatggc aactttgacg
     1621 atgctgcctg ggctcaggga cgatgtgacg ccatcgtgcg atctggcgct ggaaccgtcg
     1681 gacgaactca gattgcccga cgatccgctg ctgtctggac cgacggatgc gtggaggtcc
     1741 tggctgattc gggtaaccga cgaccctcta ctcgacctcg agctaaggag taagtgtctg
     1801 tggtatctaa gctatttatc actctttaca acttctacct caactatcta ctttaataaa
     1861 tgaatatcgt ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaatat
     1921 aacttcgtat agcaggagtt atccgaagcg agtagggata acagggtaat acggagagac
     1981 ccccaagggc gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag
     2041 agggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg
     2101 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2161 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2221 cctatacgta cggcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
     2281 tgtggatgta cagagtgata ttattgacac gccggggcga cggatggtga tccccctggc
     2341 cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg
     2401 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg
     2461 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     2521 gttctgggga atataaatgt caggcatgag attatcaaaa aggatcttca cctagatcct
     2581 tttcacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg
     2641 ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct
     2701 tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc
     2761 tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc
     2821 gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat gaggatcgtt
     2881 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     2941 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3001 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3061 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3121 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3181 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3241 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3301 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3361 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     3421 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3481 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3541 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3601 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     3661 tcttgacgag ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt attttctcct
     3721 tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg
     3781 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3841 aaccctgata aatgcttcaa taatagcacg tgaggagggc caccatggcc aagttgacca
     3901 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
     3961 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
     4021 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
     4081 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
     4141 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
     4201 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac
     4261 acgtgctaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4321 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4381 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4441 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4501 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     4561 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4621 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4681 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4741 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4801 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4861 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4921 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4981 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5041 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     5101 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     5161 cggaag
//
