LOCUS       sequence-254521-        7616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dominant marker in Cryptococcus neoformans. Targets DNA constructs 
            to a Safe Haven Site 1, 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 7616)
  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 7616)
  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: pPEE16
FEATURES             Location/Qualifiers
     source          1..7616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        800..904
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(905..973,974..1765)
                     /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(1123..1142)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1936..2524
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2425..2444
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2678..2695
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2812..2833
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2848..2853,2854..2871,2872..2878)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2886..2902
                     /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     2891..2913
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2910..2926
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2910..2926
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2922..3287
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     2945..2965
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2947..2965
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    2978..3085
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3002..3018
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3002..3018
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3052..3068)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3053..3069)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3093..3112)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3094..3112)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3122..3139)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3122..3138)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3131..3153)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     regulatory      3411..3420
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4646..6292
                     /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 acacgatgtt ttccatcagt aacatcgggg cacttgtaca tattctccca ggcagacaaa
       61 gatatgacaa gaccttcacg gccgacggtt gcaacatcct tctccaattt gagctgctac
      121 aatctcctgc ttcatggcat tccagtcaca tcaaggtatc catctctttc ggcatagcta
      181 atctaggaag aatgaatata ccacgtctat aactgatcag acctaaaggg cttcagtatc
      241 ggttatacac caggatcaat ggcaaataag actcacaaat tgggatgatc atcagcagat
      301 ggtgttattc aacgggcaga ccgtgagtac aaagtgttca gcgttgaatg gcaccgtata
      361 tacaaatatg ctgagcctgt tataacctta gcttcttctc ctgttgcttc ccttggcctg
      421 taaagtcaag ttccgccgtg ttctattatc ataaatgatc gacttccatc aatggaaggg
      481 taagctcgcg tccttaagca taggcggtct cagagtgcca tccagtccag cgctcaaggt
      541 tcctaggaag gggggttcgc ctttaagaat tcagggggct gtggtagctc gacgatgcca
      601 ggtgtcatgg agaggtccaa gaaattgatg ataaccattc cctagtccgc aaagtgcctc
      661 gtatgatcca aagtgccaag attgtacgat agggtggata tccaaagcaa gaagataaac
      721 gtccaattcc atgatttgaa gtctatgggc aggcctgccg ctacagggcg cgtcaggtgg
      781 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
      841 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      901 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
      961 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1021 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1081 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1141 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1201 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1261 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1321 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1381 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1441 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     1501 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     1561 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     1621 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     1681 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     1741 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1801 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1861 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1921 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1981 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2041 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     2101 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2161 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2221 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2281 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2341 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2401 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2461 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2521 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2581 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2641 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2701 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2761 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     2821 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     2881 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     2941 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg
     3001 ctctagaact agtggatccc ccgggctgca ggaattcgat atcaagctta tcgataccgt
     3061 cgacctcgag ggggggcccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     3121 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     3181 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     3241 gcccttccca acagttgcgc agcctgaatg gcgaatggaa attgtaagcg ttaatatttt
     3301 gttaaaattc gcgttaaatt tttgttaaat cagctcattt ttttaaccaa taggccgaaa
     3361 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt ccatggcggc
     3421 cgccattcta tgctatacgg tacaagattg tggctactat gcaagctcca tagcgaatga
     3481 tcttacactg accttgtcga ccgttctgaa tgattaacaa tggtcgcaag tgtggttgac
     3541 ccacctgcgg gcgcttctta actatctatc atcggtatgt caagtatcac accctttaaa
     3601 caaaagaata agagcataat acagcagtct catcatggca acccttcgcg caatgtacaa
     3661 aaagcagcca gaagccccca cgcacaagca ataatgtcgt gttctaccct gccatgaaca
     3721 agcgtcatgg ccattagcga acgccccaat attcagcccc attcgccaag caagttgcct
     3781 cgcccccttc ccagaagatc atgggcatct cgtcttcttc taatttcgca ttttgtgaag
     3841 ctccattcat gcacgtcggc ttctggacga ctcttatcat atgcattggc ctgctcggtg
     3901 tgaggatgag ccactcccac atgtttagac ggcaggggcc tcttgccacc cgttgaattt
     3961 ttggtgcgaa gacgtgcctg ggaacgacct ccactttaaa tttctccgtt gttgcggtcg
     4021 ctccatcctc ctcacatttc ttgcctccta agtcccatgt gccacactcg gtgaaaaacg
     4081 gagtttgata cggatgaaat ttccagtgag cttttcacat tctggaggtt tcgaaaaaaa
     4141 ttgtgggatt ttctgaatta atccgagaat ccgccctgcc catcaaccgg aaagaattaa
     4201 atccctcgca cattcctttc tcccaccacc ttctttatct ctacattctt taaccaaaac
     4261 acctaggtga gtacccaaag tcttttgaca cctttgaaaa tccgcgccag tcgtagggca
     4321 cgtaaaacaa ttctcgctgg tggcttctgc aggtgaagtc ggggcagagt ggagctgagc
     4381 atggtcgacc acccacatca ttctgtcttg tttttggcga tgtcttccat gcttttgccc
     4441 tcatctgttg cgtcgactgc agagtactgt tttgccacta ctgagctcaa catgggcttg
     4501 tgtgtggtga agaaaaaaat ttggtggctc ttgaaaattt tcaacccatc ggatacatgc
     4561 ccttccaact cacaacgccc taacacttta ttgtcctatc attgcctctt cgcacatgct
     4621 aacgatctcc acagaaaact tcaaaatgcc tcaatcctgg gaagaactgg ccgctgataa
     4681 gcgcgcccgc ctcgcaaaaa ccatccctga tgaatggaaa gtccagacgc tgcctgcgga
     4741 agacagcgtt attgatttcc caaagaaatc ggggatcctt tcagaggccg aactgaagat
     4801 cacagaggcc tccgctgcag atcttgtgtc caagctggcg gccggagagt tgacctcggt
     4861 ggaagttacg ctagcattct gtaaacgggc agcaatcgcc cagcagttaa caaactgcgc
     4921 ccacgagttc ttccctgacg ccgctctcgc gcaggcaagg gaactcgatg aatactacgc
     4981 aaagcacaag agacccgttg gtccactcca tggcctcccc atctctctca aagaccagct
     5041 tcgagtcaag ggctacgaaa catcaatggg ctacatctca tggctaaaca agtacgacga
     5101 aggggactcg gttctgacaa ccatgctccg caaagccggt gccgtcttct acgtcaagac
     5161 ctctgtcccg cagaccctga tggtctgcga gacagtcaac aacatcatcg ggcgcaccgt
     5221 caacccacgc aacaagaact ggtcgtgcgg cggcagttcc ggtggtgagg gtgcgatcgt
     5281 tgggattcgt ggtggcgtca tcggtgtagg aacggatatc ggtggctcga ttcgagtgcc
     5341 ggccgcgttc aacttcctgt acggtctaag gccgagtcat gggcggctgc cgtatgcaaa
     5401 gatggcgaac agcatggagg gtcaggagac ggtgcacagc gttgtcgggc cgattacgca
     5461 ctctgttgag gacctccgcc tcttcaccaa atccgtcctc ggtcaggagc catggaaata
     5521 cgactccaag gtcatcccca tgccctggcg ccagtccgag tcggacatta ttgcctccaa
     5581 gatcaagaac ggcgggctca atatcggcta ctacaacttc gacggcaatg tccttccaca
     5641 ccctcctatc ctgcgcggcg tggaaaccac cgtcgccgca ctcgccaaag ccggtcacac
     5701 cgtgaccccg tggacgccat acaagcacga tttcggccac gatctcatct cccatatcta
     5761 cgcggctgac ggcagcgccg acgtaatgcg cgatatcagt gcatccggcg agccggcgat
     5821 tccaaatatc aaagacctac tgaacccgaa catcaaagct gttaacatga acgagctctg
     5881 ggacacgcat ctccagaagt ggaattacca gatggagtac cttgagaaat ggcgggaggc
     5941 tgaagaaaag gccgggaagg aactggacgc catcatcgcg ccgattacgc ctaccgctgc
     6001 ggtacggcat gaccagttcc ggtactatgg gtatgcctct gtgatcaacc tgctggattt
     6061 cacgagcgtg gttgttccgg ttacctttgc ggataagaac atcgataaga agaatgagag
     6121 tttcaaggcg gttagtgagc ttgatgccct cgtgcaggaa gagtatgatc cggaggcgta
     6181 ccatggggca ccggttgcag tgcaggttat cggacggaga ctcagtgaag agaggacgtt
     6241 ggcgattgca gaggaagtgg ggaagttgct gggaaatgtg gtgactccat agaaggttga
     6301 gcgcgtaata gtgggttggt cttgtctcta tccattcatc tgtgagacat actgatttca
     6361 actttttcat cagataataa ggatggtgtg agataagcaa ggctgggtta tgaggtttgg
     6421 gactagatgg tgtttcgcgt ctaaaggtga gtcttcgttg gttcaatcac attgatctga
     6481 tcgctgatca attgtcagcc tctgaatgtg ctcttgaaga aaatggttgt ctgtaagaat
     6541 ccagaaccaa aaacggtcat ttcaagtatg tatcctccgt gaagttcctt tagctcttgc
     6601 taatattgga ctttagcgtg ctttagccaa agacagggtc gcgaggttaa gaggatgggc
     6661 caggaaaaat ctaagttaga tttgtcacac aaagaaaaag attgtctcag ttttggtttt
     6721 gtttggtttg atttatgcat aggaaatatt tatgatcgaa atcactcatt tcctgcttcg
     6781 taaatatcat ctactatatg cttcgtaaat gtccatctcc tacatattgt ggatatgata
     6841 aatgcaatcc tctcgtctac actctcggct tatgcctttg tcttctttgc gctcctttta
     6901 gccttctttt tattcctagt cttcaagccc atccgacaca agttcaaagg gctggcgctg
     6961 cttaggatcc atcacaccat tcgtcaacgt attgtgaatc aaagaaatgc tcttcgatat
     7021 tctttatcag ggcaagggaa cagatagata ccagcaggct tcacaactct gagtcgctcg
     7081 gccaaagagg aaaagaaaag aggatgccgg cactttctat acagtcacaa gacgcgaatg
     7141 acaatccggc actttatcgc atcatgtctg aatttgtgaa gggactttga gatataatca
     7201 gggggtcaga cgcactttac aactctgaat cgcttcggag taaggaagtg gaactgacag
     7261 atacacttta tagagccttg ggaaccatct ggaccaagaa gaagggaaaa aaaggctaca
     7321 gtgagtgagc aacctatgct cacctttatc ctgacgttga ttgagtctag aggccaaaga
     7381 tccaaggcaa ggcgaaatat tccattgtcc agttcatggc tgagtgaccg ttgtttaggt
     7441 tgatcgaggc gaaaagagaa gtcgatgttg atgattgtga gaccgaaatc agacttggcc
     7501 gctttttata gttcaaaaaa ggggcccaga ggtcagtggg ctcgccactc gatgcataag
     7561 caatccactt ggcacagact ggtttggttt aattaacaca agtatcgtgg cgcgcc
//
