LOCUS       sequence-267942-        8156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Detecting gene essentiality in Cryptococcus neoformans. Targets DNA 
            constructs to the Safe Haven Site 1, a small gene-free region. 
            Contains amdS2 counterselectable marker. This version of the Safe 
            Haven vector includes direct repeats, enabling the formation of 
            popouts on fluoroacetamide..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8156)
  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 8156)
  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: pPEE17
FEATURES             Location/Qualifiers
     source          1..8156
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1070..1174
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1175..1243,1244..2035)
                     /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(1393..1412)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2206..2794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2695..2714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2948..2965
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3082..3103
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3118..3123,3124..3141,3142..3148)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3156..3172
                     /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     3161..3183
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3180..3196
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3180..3196
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3192..3557
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     3215..3235
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3217..3235
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    3248..3355
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3272..3288
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3272..3288
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3322..3338)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3323..3339)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3363..3382)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3364..3382)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3392..3409)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3392..3408)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3401..3423)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     regulatory      3681..3690
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4916..6562
                     /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 aagttctact ctagccatct aatcctcaac
      781 tgccgactac tcgttctacg tattagcgca ctaatactca tgcgtctagt tagcttccat
      841 ctacgtattc gactcctgat cctaatgcgt cgccggctat cggatctcat gcgtctacga
      901 ctccgactgc agctaatcat tagcctacga ctccaggttc attaccgttg actattcgtt
      961 ttgctccatt tgactctact cactcactgc gcactcgttc taatatagtg gcatgattga
     1021 aggcctgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1081 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1141 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1201 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1261 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1321 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1381 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     1441 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1501 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     1561 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     1621 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1681 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     1741 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     1801 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     1861 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     1921 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     1981 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2041 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2101 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2161 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2221 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2281 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2341 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2401 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2461 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2521 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2581 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2641 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2701 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2761 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2821 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2881 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2941 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     3001 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     3061 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     3121 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     3181 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     3241 aaagctggag ctccaccgcg gtggcggccg ctctagaact agtggatccc ccgggctgca
     3301 ggaattcgat atcaagctta tcgataccgt cgacctcgag ggggggcccg gtacccaatt
     3361 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     3421 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     3481 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     3541 gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     3601 cagctcattt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3661 agaccgagat agggttgagt ccatggcggc cgccattcta tgctatacgg tacaagattg
     3721 tggctactat gcaagctcca tagcgaatga tcttacactg accttgtcga ccgttctgaa
     3781 tgattaacaa tggtcgcaag tgtggttgac ccacctgcgg gcgcttctta actatctatc
     3841 atcggtatgt caagtatcac accctttaaa caaaagaata agagcataat acagcagtct
     3901 catcatggca acccttcgcg caatgtacaa aaagcagcca gaagccccca cgcacaagca
     3961 ataatgtcgt gttctaccct gccatgaaca agcgtcatgg ccattagcga acgccccaat
     4021 attcagcccc attcgccaag caagttgcct cgcccccttc ccagaagatc atgggcatct
     4081 cgtcttcttc taatttcgca ttttgtgaag ctccattcat gcacgtcggc ttctggacga
     4141 ctcttatcat atgcattggc ctgctcggtg tgaggatgag ccactcccac atgtttagac
     4201 ggcaggggcc tcttgccacc cgttgaattt ttggtgcgaa gacgtgcctg ggaacgacct
     4261 ccactttaaa tttctccgtt gttgcggtcg ctccatcctc ctcacatttc ttgcctccta
     4321 agtcccatgt gccacactcg gtgaaaaacg gagtttgata cggatgaaat ttccagtgag
     4381 cttttcacat tctggaggtt tcgaaaaaaa ttgtgggatt ttctgaatta atccgagaat
     4441 ccgccctgcc catcaaccgg aaagaattaa atccctcgca cattcctttc tcccaccacc
     4501 ttctttatct ctacattctt taaccaaaac acctaggtga gtacccaaag tcttttgaca
     4561 cctttgaaaa tccgcgccag tcgtagggca cgtaaaacaa ttctcgctgg tggcttctgc
     4621 aggtgaagtc ggggcagagt ggagctgagc atggtcgacc acccacatca ttctgtcttg
     4681 tttttggcga tgtcttccat gcttttgccc tcatctgttg cgtcgactgc agagtactgt
     4741 tttgccacta ctgagctcaa catgggcttg tgtgtggtga agaaaaaaat ttggtggctc
     4801 ttgaaaattt tcaacccatc ggatacatgc ccttccaact cacaacgccc taacacttta
     4861 ttgtcctatc attgcctctt cgcacatgct aacgatctcc acagaaaact tcaaaatgcc
     4921 tcaatcctgg gaagaactgg ccgctgataa gcgcgcccgc ctcgcaaaaa ccatccctga
     4981 tgaatggaaa gtccagacgc tgcctgcgga agacagcgtt attgatttcc caaagaaatc
     5041 ggggatcctt tcagaggccg aactgaagat cacagaggcc tccgctgcag atcttgtgtc
     5101 caagctggcg gccggagagt tgacctcggt ggaagttacg ctagcattct gtaaacgggc
     5161 agcaatcgcc cagcagttaa caaactgcgc ccacgagttc ttccctgacg ccgctctcgc
     5221 gcaggcaagg gaactcgatg aatactacgc aaagcacaag agacccgttg gtccactcca
     5281 tggcctcccc atctctctca aagaccagct tcgagtcaag ggctacgaaa catcaatggg
     5341 ctacatctca tggctaaaca agtacgacga aggggactcg gttctgacaa ccatgctccg
     5401 caaagccggt gccgtcttct acgtcaagac ctctgtcccg cagaccctga tggtctgcga
     5461 gacagtcaac aacatcatcg ggcgcaccgt caacccacgc aacaagaact ggtcgtgcgg
     5521 cggcagttcc ggtggtgagg gtgcgatcgt tgggattcgt ggtggcgtca tcggtgtagg
     5581 aacggatatc ggtggctcga ttcgagtgcc ggccgcgttc aacttcctgt acggtctaag
     5641 gccgagtcat gggcggctgc cgtatgcaaa gatggcgaac agcatggagg gtcaggagac
     5701 ggtgcacagc gttgtcgggc cgattacgca ctctgttgag gacctccgcc tcttcaccaa
     5761 atccgtcctc ggtcaggagc catggaaata cgactccaag gtcatcccca tgccctggcg
     5821 ccagtccgag tcggacatta ttgcctccaa gatcaagaac ggcgggctca atatcggcta
     5881 ctacaacttc gacggcaatg tccttccaca ccctcctatc ctgcgcggcg tggaaaccac
     5941 cgtcgccgca ctcgccaaag ccggtcacac cgtgaccccg tggacgccat acaagcacga
     6001 tttcggccac gatctcatct cccatatcta cgcggctgac ggcagcgccg acgtaatgcg
     6061 cgatatcagt gcatccggcg agccggcgat tccaaatatc aaagacctac tgaacccgaa
     6121 catcaaagct gttaacatga acgagctctg ggacacgcat ctccagaagt ggaattacca
     6181 gatggagtac cttgagaaat ggcgggaggc tgaagaaaag gccgggaagg aactggacgc
     6241 catcatcgcg ccgattacgc ctaccgctgc ggtacggcat gaccagttcc ggtactatgg
     6301 gtatgcctct gtgatcaacc tgctggattt cacgagcgtg gttgttccgg ttacctttgc
     6361 ggataagaac atcgataaga agaatgagag tttcaaggcg gttagtgagc ttgatgccct
     6421 cgtgcaggaa gagtatgatc cggaggcgta ccatggggca ccggttgcag tgcaggttat
     6481 cggacggaga ctcagtgaag agaggacgtt ggcgattgca gaggaagtgg ggaagttgct
     6541 gggaaatgtg gtgactccat agaaggttga gcgcgtaata gtgggttggt cttgtctcta
     6601 tccattcatc tgtgagacat actgatttca actttttcat cagataataa ggatggtgtg
     6661 agataagcaa ggctgggtta tgaggtttgg gactagatgg tgtttcgcgt ctaaaggtga
     6721 gtcttcgttg gttcaatcac attgatctga tcgctgatca attgtcagcc tctgaatgtg
     6781 ctcttgaaga aaatggttgt ctgtaagaat ccagaaccaa aaacggtcat ttcaagtatg
     6841 tatcctccgt gaagttcctt tagctcttgc taatattgga ctttagcgtg ctttagccaa
     6901 agacagggtc gcgaggttaa gaggatgggc caggaaaaat ctaagttaga tttgtcacac
     6961 aaagaaaaag attgtctcag ttttggtttt gtttggtttg atttatgcat aggaaatatt
     7021 tatgatcgaa atcactcatt tcctgcttcg taaatatcat ctactatatg cttcgtaaat
     7081 gtccatctcc tacatattgt ggatatgata aatgcaatcc tctcgtctac actctcggct
     7141 tatgcctttg tcttctttgc gctcctttta gccttctttt tattcctagt cttcaagccc
     7201 atcaagttct actctagcca tctaatcctc aactgccgac tactcgttct acgtattagc
     7261 gcactaatac tcatgcgtct agttagcttc catctacgta ttcgactcct gatcctaatg
     7321 cgtcgccggc tatcggatct catgcgtcta cgactccgac tgcagctaat cattagccta
     7381 cgactccagg ttcattaccg ttgactattc gttttgctcc atttgactct actcactcac
     7441 tgcgcactcg ttctaatata gtggcatgat tgacgacaca agttcaaagg gctggcgctg
     7501 cttaggatcc atcacaccat tcgtcaacgt attgtgaatc aaagaaatgc tcttcgatat
     7561 tctttatcag ggcaagggaa cagacagata ccagcaggct tcacaactct gagtcgctcg
     7621 gccaaagagg aaaagaaaag aggatgccgg cactttctat acagtcacaa gacgcgaatg
     7681 acaatccggc actttatcgc atcatgtctg aatttgtgaa gggactttga gatataatca
     7741 gggggtcaga cgcactttac aactctgaat cgcttcggag taaggaagtg gaactgacag
     7801 atacacttta tagagccttg ggaaccatct ggaccaagaa gaagggaaaa aaaggctaca
     7861 gtgagtgagc aacctatgct cacctttatc ctgacgttga ttgagtctag aggccaaaga
     7921 tccaaggcaa ggcgaaatat tccattgtcc agttcatggc tgagtgaccg ttgtttaggt
     7981 tgatcgaggc gaaaagagaa gtcgatgttg atgattgtga gaccgaaatc agacttggcc
     8041 gctttttata gttcaaaaaa ggggcccaga ggtcagtggg ctcgccactc gatgcataag
     8101 caatccactt ggcacagact ggtttggttt aattaacaca agtatcgtgg cgcgcc
//
