LOCUS       sequence-347241-        6823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a G418 (NEOr) selectable marker with codon-optimization for 
            expression in CUG clade fungi. Also encodes RFP tagging of Candida 
            auris ENO1 to measure transformation efficiency..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6823)
  AUTHORS   Santana DJ, O'Meara TR
  TITLE     Forward and reverse genetic dissection of morphogenesis identifies 
            filament-competent Candida auris strains.
  JOURNAL   Nat Commun. 2021 Dec 10;12(1):7197. doi: 10.1038/s41467-021-27545-5.
  PUBMED    34893621
REFERENCE   2  (bases 1 to 6823)
  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: "Nat 
            Commun."; date: "2021-12-10"; volume: "12(1):7197. doi"; pages: " 
            10.1038/s41467-021-27545-5"
COMMENT     Sequence Label: pTO149_RFP_CauNEO
FEATURES             Location/Qualifiers
     source          1..6823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     479..498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(717..821)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     889..907
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(945..967)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1067..1086
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1280..1302
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1294..1311
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1295..1311
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3511..3854
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3868..4668
                     /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="MSMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3928..3947)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4538..4557
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      4683..4880
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(5518..5534)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5518..5534)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5531..5553)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5542..5558
                     /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)."
     promoter        complement(join(5566..5572,5573..5590,5591..5596))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5611..5632
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5749..5766)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5920..6508)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6000..6019)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6679..6823,1..647,648..716))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
       61 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
      121 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
      181 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      241 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      301 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      361 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      421 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
      481 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
      541 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
      601 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
      661 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      721 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      781 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      841 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
      901 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
      961 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     1021 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     1081 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     1141 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     1201 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     1261 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattcgag
     1321 ctcggtaccg gtgagcaatt ggctgacttg tacgtctctt tgatcgacaa gtacccaatt
     1381 gtttccattg aggacccatt cgccgaggac gactgggagt cctggtccaa gttctaccca
     1441 agagtggttg acaagatcca gattgtcggt gatgacttga ccgtcaccaa cccaatcaga
     1501 atcaagactg ccattgagaa gaaggctgcc aacgccttgt tgttgaaggt caaccagatt
     1561 ggttctttga ccgaatctat tcaggccgcc aaggactctt acgctgccga ctggggtgtt
     1621 atggtttccc acagatctgg tgagtctgag gacaccacca ttgctgactt ggttgtcggt
     1681 ttgagatctg gccagattaa gactggtgct cctgccagat ccgagagatt ggccaagctt
     1741 aaccagatct tgagaatcga ggaggagttg ggtgacaagg ctatctacgc cggtgaacac
     1801 ttcaaggctg ctcaaaactt gggtggtggt gtttcaaaag gtgaagatga taatatggct
     1861 attattaaag aatttatgag atttaaagtt catatggaag gttcagttaa tggtcatgaa
     1921 tttgaaattg aaggtgaagg tgaaggtaga ccatatgaag gtactcaaac tgctaaattg
     1981 aaagttacta aaggtggtcc attaccattt gcttgggata ttttgtcacc acaatttatg
     2041 tatggttcaa aagcttatgt taaacatcca gctgatattc cagattattt aaaattgtca
     2101 tttccagaag gttttaaatg ggaaagagtt atgaattttg aagatggtgg tgttgttact
     2161 gttactcaag attcatcatt acaagatggt gaatttattt ataaagttaa attgagaggt
     2221 actaattttc catcagatgg tccagttatg caaaaaaaaa ctatgggttg ggaagcttca
     2281 tcagaaagaa tgtatccaga agatggtgct ttaaaaggtg aaattaaaca aagattgaaa
     2341 ttaaaagatg gtggtcatta tgatgctgaa gttaaaacta cttataaagc taaaaaacca
     2401 gttcaattac caggtgctta taatgttaat attaaattgg atattacttc acataatgaa
     2461 gattatacta ttgttgaaca atatgaaaga gctgaaggta gacattcaac tggtggtatg
     2521 gatgaattat ataaataagt agcagcatga ttatgaacag gtaatacaca gttaacgaga
     2581 taagaactcg tagaagaagc catggttgac acacaacgtg tagatgaaag tcaatataaa
     2641 tacaggttct ggcccaccga ttacgacact gactatactt taagaaagcg atttgctttg
     2701 gccatcattg aacaggtact cgacagaata ccagttcgtc tcttcgatcc tcttccacag
     2761 gcaatgcatc atggagttac ttctcattgc cttggttcat cgaatcagca tcctgcgcca
     2821 ctttggccat ccaccgctgc accaacagct cattctcctt cttcaacttc tcaatctcct
     2881 cctttaacac gttctgctgg atctcatgca ccaagtgctc gtcgttgatg atctcgaccg
     2941 tccggttctt ctctgcaact tctagcacca aatggtcaat tttcaggttc aattgcgcca
     3001 ctgtcagctc caacagccgg atctttttgt cctgctgtgc cactttcccc tccagactct
     3061 cgagtcgtag cgtctgctgg ttgagtctct caataagcgt ttcgttctcc agaatcaaca
     3121 cctggcgctc cttctcggcc aacgacgcat tgccctgcgg aagaagcctt gcgagctgtg
     3181 agaatgcctt aaagtacgct ccgtcttttt tctcgagctc gtcccgctcg tggagccttt
     3241 cctcaatgag cttccatggg tccatgtagt tggtgagagt tgcaggtgtg cagggcactt
     3301 ttttggtgaa gacggtggaa ctgtgggatt tgtggtgggc aattaattga ttgatattgt
     3361 agttttgggg tttattggga gttgaaatag actcttgtga ataaagtgat tgaaatggtg
     3421 aatgtggggt ttatcaaggc gatttgtcgg ttgatgggtt tgcagtttag gatcaagctt
     3481 gcctcgtccc cgccgggtca cccggccagc gacatggagg cccagaatac cctccttgac
     3541 agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat ttagcccata
     3601 catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc gcgaagcaaa
     3661 aattacggct cctcgctgca gacctgcgag cagggaaacg ctcccctcac agacgcgttg
     3721 aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt gccactgagg
     3781 ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca catcacatcc
     3841 gaacataaac aaccggatcc tacaattatg tctatgattg aacaagatgg attgcacgca
     3901 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3961 ggctgctctg atgccgccgt gttccggctc tcagcgcagg ggcgcccggt tctttttgtc
     4021 aagaccgacc tctccggtgc cctcaatgaa ctccaggacg aggcagcgcg gctatcgtgg
     4081 ctcgccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4141 gactggctcc tattgggcga agtgccgggg caggatctcc tctcatccca ccttgctcct
     4201 gccgagaaag tatccatcat ggctgatgca atgcggcggc tccatacgct tgatccggct
     4261 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4321 gccggtcttg tcgatcagga tgatctcgac gaagagcatc aggggctcgc gccagccgaa
     4381 ctcttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4441 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4501 ggccggctcg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4561 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4621 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgata actcgagtca
     4681 gtactgacaa taaaaagatt cttgttttca agaacttgtc atttgtatag tttttttata
     4741 ttgtagttgt tctattttaa tcaaatgtta gcgtgattta tatttttttt cgcctcgaca
     4801 tcatctgccc agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt
     4861 gaatgctggt cgctatactg ctgtcgattc gatactaacg ccgccatcca gtgtttgcgc
     4921 ttcaaaccac tttggtatat aatctagacg aaatatcata ctaagaacgt tacaatgcag
     4981 tagatatgta caagactagc ttctcgaaag ctcatctaaa gcctcaacta ttctctttac
     5041 cacgactctc acatcctcaa gagtattgta caatggtgtg ggtgcaatac gaataacatc
     5101 gggtctcctc tcgtcgcaaa tgattgcatg cttgtgtaag tactcgttta cctgttccat
     5161 aacattcttg cttctgtcgt catcatgcgg ttggaataac actgaaagtt gacatcctct
     5221 ctcactttct tcacatggag tcaagatctt gaatcccaac agtgcattgt ccagcggatc
     5281 ttcgatgtag tatttgctgc ccttcaaaag ccttagcaag cagccagtca aagaaacact
     5341 agtttttcta agattatcca accctcccgc tagttcaaaa acatcaagag aactcttcaa
     5401 tgcaacacag tcaatcacca gcgggttaga ttgacgatag gagagggcag agttgattgg
     5461 gtcaaagacg gatcctctag agtcgacctg caggcatgca agcttggcgt aatcatggtc
     5521 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5581 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5641 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5701 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5761 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5821 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5881 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5941 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6001 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6061 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6121 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6181 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6241 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6301 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6361 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6421 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6481 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6541 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6601 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6661 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6721 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6781 gggcttacca tctggcccca gtgctgcaat gataccgcga gac
//
