LOCUS       sequence-254573-        6989 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recycable marker for use in Cryptococcus neoformans. Contains the 
            amdS2 counterselectable marker flanked by direct repeats..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6989)
  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 6989)
  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: pPEE8
FEATURES             Location/Qualifiers
     source          1..6989
                     /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"
     CDS             complement(1607..3253)
                     /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(4801..4821)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4801..4819)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4840..4856)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4840..4856)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4853..4875)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4864..4880
                     /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(4888..4894,4895..4912,4913..4918))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4933..4954
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5071..5088)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5242..5830)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5322..5341)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6001..6792,6793..6861))
                     /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     6624..6643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6862..6966)
                     /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 agctttcaat catgccacta tattagaacg
      721 agtgcgcagt gagtgagtag agtcaaatgg agcaaaacga atagtcaacg gtaatgaacc
      781 tggagtcgta ggctaatgat tagctgcagt cggagtcgta gacgcatgag atccgatagc
      841 cggcgacgca ttaggatcag gagtcgaata cgtagatgga agctaactag acgcatgagt
      901 attagtgcgc taatacgtag aacgagtagt cggcagttga ggattagatg gctagagtag
      961 aacttgatgg gcttgaagac taggaataaa aagaaggcta aaaggagcgc aaagaagaca
     1021 aaggcataag ccgagagtgt agacgagagg attgcattta tcatatccac aatatgtagg
     1081 agatggacat ttacgaagca tatagtagat gatatttacg aagcaggaaa tgagtgattt
     1141 cgatcataaa tatttcctat gcataaatca aaccaaacaa aaccaaaact gagacaatct
     1201 ttttctttgt gtgacaaatc taacttagat ttttcctggc ccatcctctt aacctcgcga
     1261 ccctgtcttt ggctaaagca cgctaaagtc caatattagc aagagctaaa ggaacttcac
     1321 ggaggataca tacttgaaat gaccgttttt ggttctggat tcttacagac aaccattttc
     1381 ttcaagagca cattcagagg ctgacaattg atcagcgatc agatcaatgt gattgaacca
     1441 acgaagactc acctttagac gcgaaacacc atctagtccc aaacctcata acccagcctt
     1501 gcttatctca caccatcctt attatctgat gaaaaagttg aaatcagtat gtctcacaga
     1561 tgaatggata gagacaagac caacccacta ttacgcgctc aaccttctat ggagtcacca
     1621 catttcccag caacttcccc acttcctctg caatcgccaa cgtcctctct tcactgagtc
     1681 tccgtccgat aacctgcact gcaaccggtg ccccatggta cgcctccgga tcatactctt
     1741 cctgcacgag ggcatcaagc tcactaaccg ccttgaaact ctcattcttc ttatcgatgt
     1801 tcttatccgc aaaggtaacc ggaacaacca cgctcgtgaa atccagcagg ttgatcacag
     1861 aggcataccc atagtaccgg aactggtcat gccgtaccgc agcggtaggc gtaatcggcg
     1921 cgatgatggc gtccagttcc ttcccggcct tttcttcagc ctcccgccat ttctcaaggt
     1981 actccatctg gtaattccac ttctggagat gcgtgtccca gagctcgttc atgttaacag
     2041 ctttgatgtt cgggttcagt aggtctttga tatttggaat cgccggctcg ccggatgcac
     2101 tgatatcgcg cattacgtcg gcgctgccgt cagccgcgta gatatgggag atgagatcgt
     2161 ggccgaaatc gtgcttgtat ggcgtccacg gggtcacggt gtgaccggct ttggcgagtg
     2221 cggcgacggt ggtttccacg ccgcgcagga taggagggtg tggaaggaca ttgccgtcga
     2281 agttgtagta gccgatattg agcccgccgt tcttgatctt ggaggcaata atgtccgact
     2341 cggactggcg ccagggcatg gggatgacct tggagtcgta tttccatggc tcctgaccga
     2401 ggacggattt ggtgaagagg cggaggtcct caacagagtg cgtaatcggc ccgacaacgc
     2461 tgtgcaccgt ctcctgaccc tccatgctgt tcgccatctt tgcatacggc agccgcccat
     2521 gactcggcct tagaccgtac aggaagttga acgcggccgg cactcgaatc gagccaccga
     2581 tatccgttcc tacaccgatg acgccaccac gaatcccaac gatcgcaccc tcaccaccgg
     2641 aactgccgcc gcacgaccag ttcttgttgc gtgggttgac ggtgcgcccg atgatgttgt
     2701 tgactgtctc gcagaccatc agggtctgcg ggacagaggt cttgacgtag aagacggcac
     2761 cggctttgcg gagcatggtt gtcagaaccg agtccccttc gtcgtacttg tttagccatg
     2821 agatgtagcc cattgatgtt tcgtagccct tgactcgaag ctggtctttg agagagatgg
     2881 ggaggccatg gagtggacca acgggtctct tgtgctttgc gtagtattca tcgagttccc
     2941 ttgcctgcgc gagagcggcg tcagggaaga actcgtgggc gcagtttgtt aactgctggg
     3001 cgattgctgc ccgtttacag aatgctagcg taacttccac cgaggtcaac tctccggccg
     3061 ccagcttgga cacaagatct gcagcggagg cctctgtgat cttcagttcg gcctctgaaa
     3121 ggatccccga tttctttggg aaatcaataa cgctgtcttc cgcaggcagc gtctggactt
     3181 tccattcatc agggatggtt tttgcgaggc gggcgcgctt atcagcggcc agttcttccc
     3241 aggattgagg cattttgaag ttttctgtgg agatcgttag catgtgcgaa gaggcaatga
     3301 taggacaata aagtgttagg gcgttgtgag ttggaagggc atgtatccga tgggttgaaa
     3361 attttcaaga gccaccaaat ttttttcttc accacacaca agcccatgtt gagctcagta
     3421 gtggcaaaac agtactctgc agtcgacgca acagatgagg gcaaaagcat ggaagacatc
     3481 gccaaaaaca agacagaatg atgtgggtgg tcgaccatgc tcagctccac tctgccccga
     3541 cttcacctgc agaagccacc agcgagaatt gttttacgtg ccctacgact ggcgcggatt
     3601 ttcaaaggtg tcaaaagact ttgggtactc acctaggtgt tttggttaaa gaatgtagag
     3661 ataaagaagg tggtgggaga aaggaatgtg cgagggattt aattctttcc ggttgatggg
     3721 cagggcggat tctcggatta attcagaaaa tcccacaatt tttttcgaaa cctccagaat
     3781 gtgaaaagct cactggaaat ttcatccgta tcaaactccg tttttcaccg agtgtggcac
     3841 atgggactta ggaggcaaga aatgtgagga ggatggagcg accgcaacaa cggagaaatt
     3901 taaagtggag gtcgttccca ggcacgtctt cgcaccaaaa attcaacggg tggcaagagg
     3961 cccctgccgt ctaaacatgt gggagtggct catcctcaca ccgagcaggc caatgcatat
     4021 gataagagtc gtccagaagc cgacgtgcat gaatggagct tcacaaaatg cgaaattaga
     4081 agaagacgag atgcccatga tcttctggga agggggcgag gcaacttgct tggcgaatgg
     4141 ggctgaatat tggggcgttc gctaatggcc atgacgcttg ttcatggcag ggtagaacac
     4201 gacattattg cttgtgcgtg ggggcttctg gctgcttttt gtacattgcg cgaagggttg
     4261 ccatgatgag actgctgtat tatgctctta ttcttttgtt taaagggtgt gatacttgac
     4321 ataccgatga tagatagtta agaagcgccc gcaggtgggt caaccacact tgcgaccatt
     4381 gttaatcatt cagaacggtc gacaaggtca gtgtaagatc attcgctatg gagcttgcat
     4441 agtagccaca atcttgtacc gtatagcata gaatggcggc cgctcaatca tgccactata
     4501 ttagaacgag tgcgcagtga gtgagtagag tcaaatggag caaaacgaat agtcaacggt
     4561 aatgaacctg gagtcgtagg ctaatgatta gctgcagtcg gagtcgtaga cgcatgagat
     4621 ccgatagccg gcgacgcatt aggatcagga gtcgaatacg tagatggaag ctaactagac
     4681 gcatgagtat tagtgcgcta atacgtagaa cgagtagtcg gcagttgagg attagatggc
     4741 tagagtagaa cttactagtt ctagagcgcc gccaccgcgg tggagctcca gcttttgttc
     4801 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4861 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4921 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4981 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5041 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5101 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5161 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5221 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5281 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5341 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5401 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5461 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5521 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5581 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5641 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5701 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5761 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5821 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5881 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5941 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6001 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6061 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6961 tccgcgcaca tttccccgaa aagtgccac
//
