LOCUS       sequence-254548-        7725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dominant marker in Cryptococcus neoformans. Targets DNA constructs 
            to a Safe Haven Site 4, a small gene-free region. Contains the amdS2
            counterselectable marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7725)
  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 7725)
  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: pPEE20
FEATURES             Location/Qualifiers
     source          1..7725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        808..912
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(913..981,982..1773)
                     /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     complement(1131..1150)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1944..2532
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2433..2452
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2686..2703
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2820..2841
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2856..2861,2862..2879,2880..2886)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2894..2910
                     /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     2899..2921
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2918..2934
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2918..2934
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2930..3295
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     2953..2973
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2955..2973
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    2986..3093
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3010..3026
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3010..3026
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3060..3076)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3061..3077)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3101..3120)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3102..3120)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3130..3147)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3130..3146)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3139..3161)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     regulatory      3419..3428
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4654..6300
                     /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"
ORIGIN
        1 ttcgacacaa atgcatgtgt atctccacat ccatccctca ctaaccaacc caatgtcatc
       61 atgtagctgt gactctgcca tgtgcagtca tataataaaa agttacattg ttacatctcc
      121 ctgcgtttat aggggacaca ttgactgtct gctgaatgct atctttggtg atgtaaatac
      181 cagaaacccc catatccccg ccattgttta cttcatgtat gcatattacg tctaccctga
      241 agctgcaacg aatcccaatc caaacctatg aacatgaagg cgaaaatcat agtatgaatg
      301 ttgtggggta tatggcatag cacgatcaat ctgataaata ttaacacggg ccaaacggtt
      361 gcatcgcatt ctatctactt caactgctac gtctttggtg tgtttgttgc tctacagacg
      421 acccatgttt cccagtacac aggacctcct tcattatgtc accttttgtc ttcatccttg
      481 ccttcctccc atcctcgttc cttcctctcc tgcaaaaatt cctttttaaa agaacagcct
      541 gcgcgtttgg tggacttgta atccctccaa taaaacattc aacggcatag tcgtaaacaa
      601 gaatgttcac ctcctgattt cctcacattc cttcgccagc tctgtgaggt caaagagttc
      661 gtcaatagga agacgccgac caaaataaag acttgatgtg cggtaaggtg ctgggagggc
      721 caggcttggt acgaagggct ggaagagagt atttaaatag gcctgccgct acagggcgcg
      781 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
      841 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
      901 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
      961 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     1021 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     1081 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     1141 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     1201 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     1261 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     1321 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     1381 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     1441 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     1501 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     1561 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     1621 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     1681 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     1741 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     1801 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     1861 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     1921 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     1981 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2041 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     2101 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2161 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2221 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2281 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2341 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2401 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2461 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2521 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2581 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2641 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     2701 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     2761 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     2821 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     2881 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     2941 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct ccaccgcggt
     3001 ggcggccgct ctagaactag tggatccccc gggctgcagg aattcgatat caagcttatc
     3061 gataccgtcg acctcgaggg ggggcccggt acccaattcg ccctatagtg agtcgtatta
     3121 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     3181 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     3241 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt
     3301 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt ttaaccaata
     3361 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtcc
     3421 atggcggccg ccattctatg ctatacggta caagattgtg gctactatgc aagctccata
     3481 gcgaatgatc ttacactgac cttgtcgacc gttctgaatg attaacaatg gtcgcaagtg
     3541 tggttgaccc acctgcgggc gcttcttaac tatctatcat cggtatgtca agtatcacac
     3601 cctttaaaca aaagaataag agcataatac agcagtctca tcatggcaac ccttcgcgca
     3661 atgtacaaaa agcagccaga agcccccacg cacaagcaat aatgtcgtgt tctaccctgc
     3721 catgaacaag cgtcatggcc attagcgaac gccccaatat tcagccccat tcgccaagca
     3781 agttgcctcg cccccttccc agaagatcat gggcatctcg tcttcttcta atttcgcatt
     3841 ttgtgaagct ccattcatgc acgtcggctt ctggacgact cttatcatat gcattggcct
     3901 gctcggtgtg aggatgagcc actcccacat gtttagacgg caggggcctc ttgccacccg
     3961 ttgaattttt ggtgcgaaga cgtgcctggg aacgacctcc actttaaatt tctccgttgt
     4021 tgcggtcgct ccatcctcct cacatttctt gcctcctaag tcccatgtgc cacactcggt
     4081 gaaaaacgga gtttgatacg gatgaaattt ccagtgagct tttcacattc tggaggtttc
     4141 gaaaaaaatt gtgggatttt ctgaattaat ccgagaatcc gccctgccca tcaaccggaa
     4201 agaattaaat ccctcgcaca ttcctttctc ccaccacctt ctttatctct acattcttta
     4261 accaaaacac ctaggtgagt acccaaagtc ttttgacacc tttgaaaatc cgcgccagtc
     4321 gtagggcacg taaaacaatt ctcgctggtg gcttctgcag gtgaagtcgg ggcagagtgg
     4381 agctgagcat ggtcgaccac ccacatcatt ctgtcttgtt tttggcgatg tcttccatgc
     4441 ttttgccctc atctgttgcg tcgactgcag agtactgttt tgccactact gagctcaaca
     4501 tgggcttgtg tgtggtgaag aaaaaaattt ggtggctctt gaaaattttc aacccatcgg
     4561 atacatgccc ttccaactca caacgcccta acactttatt gtcctatcat tgcctcttcg
     4621 cacatgctaa cgatctccac agaaaacttc aaaatgcctc aatcctggga agaactggcc
     4681 gctgataagc gcgcccgcct cgcaaaaacc atccctgatg aatggaaagt ccagacgctg
     4741 cctgcggaag acagcgttat tgatttccca aagaaatcgg ggatcctttc agaggccgaa
     4801 ctgaagatca cagaggcctc cgctgcagat cttgtgtcca agctggcggc cggagagttg
     4861 acctcggtgg aagttacgct agcattctgt aaacgggcag caatcgccca gcagttaaca
     4921 aactgcgccc acgagttctt ccctgacgcc gctctcgcgc aggcaaggga actcgatgaa
     4981 tactacgcaa agcacaagag acccgttggt ccactccatg gcctccccat ctctctcaaa
     5041 gaccagcttc gagtcaaggg ctacgaaaca tcaatgggct acatctcatg gctaaacaag
     5101 tacgacgaag gggactcggt tctgacaacc atgctccgca aagccggtgc cgtcttctac
     5161 gtcaagacct ctgtcccgca gaccctgatg gtctgcgaga cagtcaacaa catcatcggg
     5221 cgcaccgtca acccacgcaa caagaactgg tcgtgcggcg gcagttccgg tggtgagggt
     5281 gcgatcgttg ggattcgtgg tggcgtcatc ggtgtaggaa cggatatcgg tggctcgatt
     5341 cgagtgccgg ccgcgttcaa cttcctgtac ggtctaaggc cgagtcatgg gcggctgccg
     5401 tatgcaaaga tggcgaacag catggagggt caggagacgg tgcacagcgt tgtcgggccg
     5461 attacgcact ctgttgagga cctccgcctc ttcaccaaat ccgtcctcgg tcaggagcca
     5521 tggaaatacg actccaaggt catccccatg ccctggcgcc agtccgagtc ggacattatt
     5581 gcctccaaga tcaagaacgg cgggctcaat atcggctact acaacttcga cggcaatgtc
     5641 cttccacacc ctcctatcct gcgcggcgtg gaaaccaccg tcgccgcact cgccaaagcc
     5701 ggtcacaccg tgaccccgtg gacgccatac aagcacgatt tcggccacga tctcatctcc
     5761 catatctacg cggctgacgg cagcgccgac gtaatgcgcg atatcagtgc atccggcgag
     5821 ccggcgattc caaatatcaa agacctactg aacccgaaca tcaaagctgt taacatgaac
     5881 gagctctggg acacgcatct ccagaagtgg aattaccaga tggagtacct tgagaaatgg
     5941 cgggaggctg aagaaaaggc cgggaaggaa ctggacgcca tcatcgcgcc gattacgcct
     6001 accgctgcgg tacggcatga ccagttccgg tactatgggt atgcctctgt gatcaacctg
     6061 ctggatttca cgagcgtggt tgttccggtt acctttgcgg ataagaacat cgataagaag
     6121 aatgagagtt tcaaggcggt tagtgagctt gatgccctcg tgcaggaaga gtatgatccg
     6181 gaggcgtacc atggggcacc ggttgcagtg caggttatcg gacggagact cagtgaagag
     6241 aggacgttgg cgattgcaga ggaagtgggg aagttgctgg gaaatgtggt gactccatag
     6301 aaggttgagc gcgtaatagt gggttggtct tgtctctatc cattcatctg tgagacatac
     6361 tgatttcaac tttttcatca gataataagg atggtgtgag ataagcaagg ctgggttatg
     6421 aggtttggga ctagatggtg tttcgcgtct aaaggtgagt cttcgttggt tcaatcacat
     6481 tgatctgatc gctgatcaat tgtcagcctc tgaatgtgct cttgaagaaa atggttgtct
     6541 gtaagaatcc agaaccaaaa acggtcattt caagtatgta tcctccgtga agttccttta
     6601 gctcttgcta atattggact ttagcgtgct ttagccaaag acagggtcgc gaggttaaga
     6661 ggatgggcca ggaaaaatct aagttagatt tgtcacacaa agaaaaagat tgtctcagtt
     6721 ttggttttgt ttggtttgat ttatgcatag gaaatattta tgatcgaaat cactcatttc
     6781 ctgcttcgta aatatcatct actatatgct tcgtaaatgt ccatctccta catattgtgg
     6841 atatgataaa tgcaatcctc tcgtctacac tctcggctta tgcctttgtc ttctttgcgc
     6901 tccttttagc cttcttttta ttcctagtct tcaagcccat ctacgtaaag ggggttaacg
     6961 agagcattta gtctagaagg gaacaaggga cttaccaatc agcaacaatc acagctatcc
     7021 ctttctccta atgccatttc aaggtatact tctcatgcgc aacgccgcaa ctcccattaa
     7081 cttctcttcc tcaactgatc cctcttctcc tcactcatct tcaataaccc gtccgtaaaa
     7141 ccggttctta caatgaggat atctcctggc tgaatgttga cttgtgcagt gtcattcttc
     7201 ggcacatgca atgagggtgc agagtgaagg ctcgttggct cgtgttaatc cacgcacgcc
     7261 gcaaggtagg caacgaaagc cgtagctgct attactattt tcatctagga cgcttccctt
     7321 cgataactcc cgttctcatt cttccctctc tctctacctt cccagtactc accgtctcct
     7381 ccattcatgc aattcctgct gtgaaaaccc tcgctccgcc ctcacttccc taatggcccc
     7441 ttccgaagta agaaaagcta tttccgactc ctcactctca cccgcatcat atcgagccct
     7501 ccgcgaggat tagaaattcc ctcgtgacga ccccattgca tacggcggcc accttaaaaa
     7561 caaagaccga ccatatcccc tctctctcca tccctttcag tacgatacca accaccatat
     7621 gattcctttc attagattgt cgtctttcct ccccgtcgca cgaattcctt ccacgatttg
     7681 actgccgcca ccgttaatta attaacacaa gtatcgtggc gcgcc
//
