LOCUS       sequence-343636-        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     478..497
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(716..820)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     888..906
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(944..966)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1066..1085
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1279..1301
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1293..1310
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1294..1310
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3510..3853
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3867..4667
                     /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(3927..3946)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4537..4556
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      4682..4879
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(5517..5533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5517..5533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5530..5552)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5541..5557
                     /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(5565..5571,5572..5589,5590..5595))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5610..5631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5748..5765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5919..6507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5999..6018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6678..6823,1..646,647..715))
                     /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 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
       61 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
      121 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
      181 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
      241 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
      301 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
      361 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
      421 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
      481 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
      541 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
      601 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
      661 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
      721 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
      781 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
      841 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
      901 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
      961 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     1021 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     1081 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     1141 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     1201 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     1261 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcgagc
     1321 tcggtaccgg tgagcaattg gctgacttgt acgtctcttt gatcgacaag tacccaattg
     1381 tttccattga ggacccattc gccgaggacg actgggagtc ctggtccaag ttctacccaa
     1441 gagtggttga caagatccag attgtcggtg atgacttgac cgtcaccaac ccaatcagaa
     1501 tcaagactgc cattgagaag aaggctgcca acgccttgtt gttgaaggtc aaccagattg
     1561 gttctttgac cgaatctatt caggccgcca aggactctta cgctgccgac tggggtgtta
     1621 tggtttccca cagatctggt gagtctgagg acaccaccat tgctgacttg gttgtcggtt
     1681 tgagatctgg ccagattaag actggtgctc ctgccagatc cgagagattg gccaagctta
     1741 accagatctt gagaatcgag gaggagttgg gtgacaaggc tatctacgcc ggtgaacact
     1801 tcaaggctgc tcaaaacttg ggtggtggtg tttcaaaagg tgaagatgat aatatggcta
     1861 ttattaaaga atttatgaga tttaaagttc atatggaagg ttcagttaat ggtcatgaat
     1921 ttgaaattga aggtgaaggt gaaggtagac catatgaagg tactcaaact gctaaattga
     1981 aagttactaa aggtggtcca ttaccatttg cttgggatat tttgtcacca caatttatgt
     2041 atggttcaaa agcttatgtt aaacatccag ctgatattcc agattattta aaattgtcat
     2101 ttccagaagg ttttaaatgg gaaagagtta tgaattttga agatggtggt gttgttactg
     2161 ttactcaaga ttcatcatta caagatggtg aatttattta taaagttaaa ttgagaggta
     2221 ctaattttcc atcagatggt ccagttatgc aaaaaaaaac tatgggttgg gaagcttcat
     2281 cagaaagaat gtatccagaa gatggtgctt taaaaggtga aattaaacaa agattgaaat
     2341 taaaagatgg tggtcattat gatgctgaag ttaaaactac ttataaagct aaaaaaccag
     2401 ttcaattacc aggtgcttat aatgttaata ttaaattgga tattacttca cataatgaag
     2461 attatactat tgttgaacaa tatgaaagag ctgaaggtag acattcaact ggtggtatgg
     2521 atgaattata taaataagta gcagcatgat tatgaacagg taatacacag ttaacgagat
     2581 aagaactcgt agaagaagcc atggttgaca cacaacgtgt agatgaaagt caatataaat
     2641 acaggttctg gcccaccgat tacgacactg actatacttt aagaaagcga tttgctttgg
     2701 ccatcattga acaggtactc gacagaatac cagttcgtct cttcgatcct cttccacagg
     2761 caatgcatca tggagttact tctcattgcc ttggttcatc gaatcagcat cctgcgccac
     2821 tttggccatc caccgctgca ccaacagctc attctccttc ttcaacttct caatctcctc
     2881 ctttaacacg ttctgctgga tctcatgcac caagtgctcg tcgttgatga tctcgaccgt
     2941 ccggttcttc tctgcaactt ctagcaccaa atggtcaatt ttcaggttca attgcgccac
     3001 tgtcagctcc aacagccgga tctttttgtc ctgctgtgcc actttcccct ccagactctc
     3061 gagtcgtagc gtctgctggt tgagtctctc aataagcgtt tcgttctcca gaatcaacac
     3121 ctggcgctcc ttctcggcca acgacgcatt gccctgcgga agaagccttg cgagctgtga
     3181 gaatgcctta aagtacgctc cgtctttttt ctcgagctcg tcccgctcgt ggagcctttc
     3241 ctcaatgagc ttccatgggt ccatgtagtt ggtgagagtt gcaggtgtgc agggcacttt
     3301 tttggtgaag acggtggaac tgtgggattt gtggtgggca attaattgat tgatattgta
     3361 gttttggggt ttattgggag ttgaaataga ctcttgtgaa taaagtgatt gaaatggtga
     3421 atgtggggtt tatcaaggcg atttgtcggt tgatgggttt gcagtttagg atcaagcttg
     3481 cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc ctccttgaca
     3541 gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt tagcccatac
     3601 atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg cgaagcaaaa
     3661 attacggctc ctcgctgcag acctgcgagc agggaaacgc tcccctcaca gacgcgttga
     3721 attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg ccactgaggt
     3781 tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac atcacatccg
     3841 aacataaaca accggatcct acaattatgt ctatgattga acaagatgga ttgcacgcag
     3901 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3961 gctgctctga tgccgccgtg ttccggctct cagcgcaggg gcgcccggtt ctttttgtca
     4021 agaccgacct ctccggtgcc ctcaatgaac tccaggacga ggcagcgcgg ctatcgtggc
     4081 tcgccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4141 actggctcct attgggcgaa gtgccggggc aggatctcct ctcatcccac cttgctcctg
     4201 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct ccatacgctt gatccggcta
     4261 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4321 ccggtcttgt cgatcaggat gatctcgacg aagagcatca ggggctcgcg ccagccgaac
     4381 tcttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4441 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4501 gccggctcgg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4561 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4621 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgataa ctcgagtcag
     4681 tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat
     4741 tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat
     4801 catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg
     4861 aatgctggtc gctatactgc tgtcgattcg atactaacgc cgccatccag tgtttgcgct
     4921 tcaaaccact ttggtatata atctagacga aatatcatac taagaacgtt acaatgcagt
     4981 agatatgtac aagactagct tctcgaaagc tcatctaaag cctcaactat tctctttacc
     5041 acgactctca catcctcaag agtattgtac aatggtgtgg gtgcaatacg aataacatcg
     5101 ggtctcctct cgtcgcaaat gattgcatgc ttgtgtaagt actcgtttac ctgttccata
     5161 acattcttgc ttctgtcgtc atcatgcggt tggaataaca ctgaaagttg acatcctctc
     5221 tcactttctt cacatggagt caagatcttg aatcccaaca gtgcattgtc cagcggatct
     5281 tcgatgtagt atttgctgcc cttcaaaagc cttagcaagc agccagtcaa agaaacacta
     5341 gtttttctaa gattatccaa ccctcccgct agttcaaaaa catcaagaga actcttcaat
     5401 gcaacacagt caatcaccag cgggttagat tgacgatagg agagggcaga gttgattggg
     5461 tcaaagacgg atcctctaga gtcgacctgc aggcatgcaa gcttggcgta atcatggtca
     5521 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5581 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5641 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5701 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5761 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5821 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5881 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5941 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6001 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6061 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6121 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6181 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6241 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6301 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6361 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6421 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6481 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6541 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6601 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6661 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6721 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6781 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acc
//
