LOCUS       sequence-257614-        7522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recycable marker for use in Cryptococcus neoformans. Contains the 
            amdS2 counterselectable marker flanked by the mCherry ORF and a 
            flexable linker at the 5' end. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7522)
  AUTHORS   Erpf PE, Stephenson CJ, Fraser JA
  TITLE     amdS as a dominant recyclable marker in Cryptococcus neoformans.
  JOURNAL   Fungal Genet Biol. 2019 Jun 17;131:103241. doi: 
            10.1016/j.fgb.2019.103241.
  PUBMED    31220607
REFERENCE   2  (bases 1 to 7522)
  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: "Fungal 
            Genet Biol."; date: "2019-06-17"; volume: "131:103241. doi"; pages: 
            " 10.1016/j.fgb.2019.103241"
COMMENT     Sequence Label: pPEE13
FEATURES             Location/Qualifiers
     source          1..7522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..460
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     586..608
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     600..617
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     601..617
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     627..646
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        627..645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     670..686
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     671..687
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(865..883)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             2461..4107
                     /codon_start=1
                     /gene="amdS"
                     /product="acetamidase from Aspergillus nidulans"
                     /label=amdS
                     /note="confers ability to process acetamide for selection 
                     in fungal cells"
                     /translation="MPQSWEELAADKRARLAKTIPDEWKVQTLPAEDSVIDFPKKSGIL
                     SEAELKITEASAADLVSKLAAGELTSVEVTLAFCKRAAIAQQLTNCAHEFFPDAALAQA
                     RELDEYYAKHKRPVGPLHGLPISLKDQLRVKGYETSMGYISWLNKYDEGDSVLTTMLRK
                     AGAVFYVKTSVPQTLMVCETVNNIIGRTVNPRNKNWSCGGSSGGEGAIVGIRGGVIGVG
                     TDIGGSIRVPAAFNFLYGLRPSHGRLPYAKMANSMEGQETVHSVVGPITHSVEDLRLFT
                     KSVLGQEPWKYDSKVIPMPWRQSESDIIASKIKNGGLNIGYYNFDGNVLPHPPILRGVE
                     TTVAALAKAGHTVTPWTPYKHDFGHDLISHIYAADGSADVMRDISASGEPAIPNIKDLL
                     NPNIKAVNMNELWDTHLQKWNYQMEYLEKWREAEEKAGKELDAIIAPITPTAAVRHDQF
                     RYYGYASVINLLDFTSVVVPVTFADKNIDKKNESFKAVSELDALVQEEYDPEAYHGAPV
                     AVQVIGRRLSEERTLAIAEEVGKLLGNVVTP"
     primer_bind     complement(5281..5297)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5281..5297)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5334..5354)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5334..5352)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5373..5389)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5373..5389)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5386..5408)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5397..5413
                     /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(5421..5427,5428..5445,5446..5451))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5466..5487
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5604..5621)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5775..6363)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5855..5874)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6534..7325,7326..7394))
                     /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"
     primer_bind     7157..7176
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7395..7499)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa atgagctgat ttaacaaaaa
      421 tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc
      481 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      541 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      601 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg
      661 ggccccccct cgaggtcgac ggtatcgata agcttggttc tggcggtggt ggatccggca
      721 tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
      781 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
      841 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
      901 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
      961 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     1021 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     1081 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     1141 tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     1201 ccctgaaggg cgagatcaag cagaggctga gcggccgcca ttctatgcta tacggtacaa
     1261 gattgtggct actatgcaag ctccatagcg aatgatctta cactgacctt gtcgaccgtt
     1321 ctgaatgatt aacaatggtc gcaagtgtgg ttgacccacc tgcgggcgct tcttaactat
     1381 ctatcatcgg tatgtcaagt atcacaccct ttaaacaaaa gaataagagc ataatacagc
     1441 agtctcatca tggcaaccct tcgcgcaatg tacaaaaagc agccagaagc ccccacgcac
     1501 aagcaataat gtcgtgttct accctgccat gaacaagcgt catggccatt agcgaacgcc
     1561 ccaatattca gccccattcg ccaagcaagt tgcctcgccc ccttcccaga agatcatggg
     1621 catctcgtct tcttctaatt tcgcattttg tgaagctcca ttcatgcacg tcggcttctg
     1681 gacgactctt atcatatgca ttggcctgct cggtgtgagg atgagccact cccacatgtt
     1741 tagacggcag gggcctcttg ccacccgttg aatttttggt gcgaagacgt gcctgggaac
     1801 gacctccact ttaaatttct ccgttgttgc ggtcgctcca tcctcctcac atttcttgcc
     1861 tcctaagtcc catgtgccac actcggtgaa aaacggagtt tgatacggat gaaatttcca
     1921 gtgagctttt cacattctgg aggtttcgaa aaaaattgtg ggattttctg aattaatccg
     1981 agaatccgcc ctgcccatca accggaaaga attaaatccc tcgcacattc ctttctccca
     2041 ccaccttctt tatctctaca ttctttaacc aaaacaccta ggtgagtacc caaagtcttt
     2101 tgacaccttt gaaaatccgc gccagtcgta gggcacgtaa aacaattctc gctggtggct
     2161 tctgcaggtg aagtcggggc agagtggagc tgagcatggt cgaccaccca catcattctg
     2221 tcttgttttt ggcgatgtct tccatgcttt tgccctcatc tgttgcgtcg actgcagagt
     2281 actgttttgc cactactgag ctcaacatgg gcttgtgtgt ggtgaagaaa aaaatttggt
     2341 ggctcttgaa aattttcaac ccatcggata catgcccttc caactcacaa cgccctaaca
     2401 ctttattgtc ctatcattgc ctcttcgcac atgctaacga tctccacaga aaacttcaaa
     2461 atgcctcaat cctgggaaga actggccgct gataagcgcg cccgcctcgc aaaaaccatc
     2521 cctgatgaat ggaaagtcca gacgctgcct gcggaagaca gcgttattga tttcccaaag
     2581 aaatcgggga tcctttcaga ggccgaactg aagatcacag aggcctccgc tgcagatctt
     2641 gtgtccaagc tggcggccgg agagttgacc tcggtggaag ttacgctagc attctgtaaa
     2701 cgggcagcaa tcgcccagca gttaacaaac tgcgcccacg agttcttccc tgacgccgct
     2761 ctcgcgcagg caagggaact cgatgaatac tacgcaaagc acaagagacc cgttggtcca
     2821 ctccatggcc tccccatctc tctcaaagac cagcttcgag tcaagggcta cgaaacatca
     2881 atgggctaca tctcatggct aaacaagtac gacgaagggg actcggttct gacaaccatg
     2941 ctccgcaaag ccggtgccgt cttctacgtc aagacctctg tcccgcagac cctgatggtc
     3001 tgcgagacag tcaacaacat catcgggcgc accgtcaacc cacgcaacaa gaactggtcg
     3061 tgcggcggca gttccggtgg tgagggtgcg atcgttggga ttcgtggtgg cgtcatcggt
     3121 gtaggaacgg atatcggtgg ctcgattcga gtgccggccg cgttcaactt cctgtacggt
     3181 ctaaggccga gtcatgggcg gctgccgtat gcaaagatgg cgaacagcat ggagggtcag
     3241 gagacggtgc acagcgttgt cgggccgatt acgcactctg ttgaggacct ccgcctcttc
     3301 accaaatccg tcctcggtca ggagccatgg aaatacgact ccaaggtcat ccccatgccc
     3361 tggcgccagt ccgagtcgga cattattgcc tccaagatca agaacggcgg gctcaatatc
     3421 ggctactaca acttcgacgg caatgtcctt ccacaccctc ctatcctgcg cggcgtggaa
     3481 accaccgtcg ccgcactcgc caaagccggt cacaccgtga ccccgtggac gccatacaag
     3541 cacgatttcg gccacgatct catctcccat atctacgcgg ctgacggcag cgccgacgta
     3601 atgcgcgata tcagtgcatc cggcgagccg gcgattccaa atatcaaaga cctactgaac
     3661 ccgaacatca aagctgttaa catgaacgag ctctgggaca cgcatctcca gaagtggaat
     3721 taccagatgg agtaccttga gaaatggcgg gaggctgaag aaaaggccgg gaaggaactg
     3781 gacgccatca tcgcgccgat tacgcctacc gctgcggtac ggcatgacca gttccggtac
     3841 tatgggtatg cctctgtgat caacctgctg gatttcacga gcgtggttgt tccggttacc
     3901 tttgcggata agaacatcga taagaagaat gagagtttca aggcggttag tgagcttgat
     3961 gccctcgtgc aggaagagta tgatccggag gcgtaccatg gggcaccggt tgcagtgcag
     4021 gttatcggac ggagactcag tgaagagagg acgttggcga ttgcagagga agtggggaag
     4081 ttgctgggaa atgtggtgac tccatagaag gttgagcgcg taatagtggg ttggtcttgt
     4141 ctctatccat tcatctgtga gacatactga tttcaacttt ttcatcagat aataaggatg
     4201 gtgtgagata agcaaggctg ggttatgagg tttgggacta gatggtgttt cgcgtctaaa
     4261 ggtgagtctt cgttggttca atcacattga tctgatcgct gatcaattgt cagcctctga
     4321 atgtgctctt gaagaaaatg gttgtctgta agaatccaga accaaaaacg gtcatttcaa
     4381 gtatgtatcc tccgtgaagt tcctttagct cttgctaata ttggacttta gcgtgcttta
     4441 gccaaagaca gggtcgcgag gttaagagga tgggccagga aaaatctaag ttagatttgt
     4501 cacacaaaga aaaagattgt ctcagttttg gttttgtttg gtttgattta tgcataggaa
     4561 atatttatga tcgaaatcac tcatttcctg cttcgtaaat atcatctact atatgcttcg
     4621 taaatgtcca tctcctacat attgtggata tgataaatgc aatcctctcg tctacactct
     4681 cggcttatgc ctttgtcttc tttgcgctcc ttttagcctt ctttttattc ctagtcttca
     4741 agcccatctc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     4801 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     4861 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     4921 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     4981 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     5041 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     5101 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     5161 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     5221 gccgccactc caccggcggc atggacgagc tgtacaagta agaattcctg cagcccgggg
     5281 gatccactag ttctagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag
     5341 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5401 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5461 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5521 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5581 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5641 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5701 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5761 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5821 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5881 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5941 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6001 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6061 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6121 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6181 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6241 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6301 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6361 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6421 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6481 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6541 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6601 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6661 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6721 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6781 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6841 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6901 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6961 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7021 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7081 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7141 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7201 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7261 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7321 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7381 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7441 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7501 acatttcccc gaaaagtgcc ac
//
