LOCUS Exported 7491 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Recyclable marker for use in Cryptococcus neoformans. Contains the amdS2 counterselectable marker flanked by the mTurquoise2 ORF and a flexible linker at the 5' end.. ACCESSION . VERSION . KEYWORDS pPEE45 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7491) AUTHORS Spencer GWK, Chua SMH, Erpf PE, Wizrah MSI, Dyba TG, Condon ND, Fraser JA TITLE Broadening the spectrum of fluorescent protein tools for use in the encapsulated human fungal pathogen Cryptococcus neoformans. JOURNAL Fungal Genet Biol. 2020 May;138:103365. doi: 10.1016/j.fgb.2020.103365. Epub 2020 Mar 4. PUBMED 32145317 REFERENCE 2 (bases 1 to 7491) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7491 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 24..128 /gene="bla" /label=AmpR promoter CDS 129..989 /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(347..366) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1160..1748 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1649..1668 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1902..1919 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2036..2057 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2072..2102 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2110..2126 /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 2115..2137 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2134..2150 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2134..2150 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 2169..2189 /label=T3 /note="T3 promoter, forward primer" promoter 2171..2189 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" CDS 3981..5627 /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(6805..6821) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6806..6822) /label=pBluescriptKS /note="For pBluescript vector" primer_bind complement(6846..6865) /label=T7 /note="T7 promoter, forward primer" promoter complement(6847..6865) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6875..6892) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6875..6891) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6884..6906) /label=M13/pUC Forward /note="In lacZ gene" rep_origin complement(7032..7488) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7170..7191) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7382..7401 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2221 gccgctctag atggtctcca agggcgagga gctcttcacc ggcgtcgtcc ccatcctcgt 2281 cgagctcgac ggcgacgtta acggccacaa gttctccgtc tccggcgagg gcgagggcga 2341 cgccacctac ggcaagctca ccctcaagtt catctgcacc accggcaagc tccccgtccc 2401 ctggcccacc ctcgtcacca ccctctcctg gggcgtccag tgcttcgccc gttaccccga 2461 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gagggctacg tccaggagcg 2521 taccatcttc ttcaaggacg acggcaacta caagacccgt gccgaggtca agttcgaggg 2581 cgacaccctc gtcaaccgta tcgagctcaa gggcatcgac ttcaaggagg acggcaacat 2641 cctcggccac aagctcgagt acaactactt ctccgacaac gtctacatca ccgccgacaa 2701 gcagaagaac ggcatcaagg ccaacttcaa gatccgtcac aacatcgagg gcggccgcca 2761 ttctatgcta tacggtacaa gattgtggct actatgcaag ctccatagcg aatgatctta 2821 cactgacctt gtcgaccgtt ctgaatgatt aacaatggtc gcaagtgtgg ttgacccacc 2881 tgcgggcgct tcttaactat ctatcatcgg tatgtcaagt atcacaccct ttaaacaaaa 2941 gaataagagc ataatacagc agtctcatca tggcaaccct tcgcgcaatg tacaaaaagc 3001 agccagaagc ccccacgcac aagcaataat gtcgtgttct accctgccat gaacaagcgt 3061 catggccatt agcgaacgcc ccaatattca gccccattcg ccaagcaagt tgcctcgccc 3121 ccttcccaga agatcatggg catctcgtct tcttctaatt tcgcattttg tgaagctcca 3181 ttcatgcacg tcggcttctg gacgactctt atcatatgca ttggcctgct cggtgtgagg 3241 atgagccact cccacatgtt tagacggcag gggcctcttg ccacccgttg aatttttggt 3301 gcgaagacgt gcctgggaac gacctccact ttaaatttct ccgttgttgc ggtcgctcca 3361 tcctcctcac atttcttgcc tcctaagtcc catgtgccac actcggtgaa aaacggagtt 3421 tgatacggat gaaatttcca gtgagctttt cacattctgg aggtttcgaa aaaaattgtg 3481 ggattttctg aattaatccg agaatccgcc ctgcccatca accggaaaga attaaatccc 3541 tcgcacattc ctttctccca ccaccttctt tatctctaca ttctttaacc aaaacaccta 3601 ggtgagtacc caaagtcttt tgacaccttt gaaaatccgc gccagtcgta gggcacgtaa 3661 aacaattctc gctggtggct tctgcaggtg aagtcggggc agagtggagc tgagcatggt 3721 cgaccaccca catcattctg tcttgttttt ggcgatgtct tccatgcttt tgccctcatc 3781 tgttgcgtcg actgcagagt actgttttgc cactactgag ctcaacatgg gcttgtgtgt 3841 ggtgaagaaa aaaatttggt ggctcttgaa aattttcaac ccatcggata catgcccttc 3901 caactcacaa cgccctaaca ctttattgtc ctatcattgc ctcttcgcac atgctaacga 3961 tctccacaga aaacttcaaa atgcctcaat cctgggaaga actggccgct gataagcgcg 4021 cccgcctcgc aaaaaccatc cctgatgaat ggaaagtcca gacgctgcct gcggaagaca 4081 gcgttattga tttcccaaag aaatcgggga tcctttcaga ggccgaactg aagatcacag 4141 aggcctccgc tgcagatctt gtgtccaagc tggcggccgg agagttgacc tcggtggaag 4201 ttacgctagc attctgtaaa cgggcagcaa tcgcccagca gttaacaaac tgcgcccacg 4261 agttcttccc tgacgccgct ctcgcgcagg caagggaact cgatgaatac tacgcaaagc 4321 acaagagacc cgttggtcca ctccatggcc tccccatctc tctcaaagac cagcttcgag 4381 tcaagggcta cgaaacatca atgggctaca tctcatggct aaacaagtac gacgaagggg 4441 actcggttct gacaaccatg ctccgcaaag ccggtgccgt cttctacgtc aagacctctg 4501 tcccgcagac cctgatggtc tgcgagacag tcaacaacat catcgggcgc accgtcaacc 4561 cacgcaacaa gaactggtcg tgcggcggca gttccggtgg tgagggtgcg atcgttggga 4621 ttcgtggtgg cgtcatcggt gtaggaacgg atatcggtgg ctcgattcga gtgccggccg 4681 cgttcaactt cctgtacggt ctaaggccga gtcatgggcg gctgccgtat gcaaagatgg 4741 cgaacagcat ggagggtcag gagacggtgc acagcgttgt cgggccgatt acgcactctg 4801 ttgaggacct ccgcctcttc accaaatccg tcctcggtca ggagccatgg aaatacgact 4861 ccaaggtcat ccccatgccc tggcgccagt ccgagtcgga cattattgcc tccaagatca 4921 agaacggcgg gctcaatatc ggctactaca acttcgacgg caatgtcctt ccacaccctc 4981 ctatcctgcg cggcgtggaa accaccgtcg ccgcactcgc caaagccggt cacaccgtga 5041 ccccgtggac gccatacaag cacgatttcg gccacgatct catctcccat atctacgcgg 5101 ctgacggcag cgccgacgta atgcgcgata tcagtgcatc cggcgagccg gcgattccaa 5161 atatcaaaga cctactgaac ccgaacatca aagctgttaa catgaacgag ctctgggaca 5221 cgcatctcca gaagtggaat taccagatgg agtaccttga gaaatggcgg gaggctgaag 5281 aaaaggccgg gaaggaactg gacgccatca tcgcgccgat tacgcctacc gctgcggtac 5341 ggcatgacca gttccggtac tatgggtatg cctctgtgat caacctgctg gatttcacga 5401 gcgtggttgt tccggttacc tttgcggata agaacatcga taagaagaat gagagtttca 5461 aggcggttag tgagcttgat gccctcgtgc aggaagagta tgatccggag gcgtaccatg 5521 gggcaccggt tgcagtgcag gttatcggac ggagactcag tgaagagagg acgttggcga 5581 ttgcagagga agtggggaag ttgctgggaa atgtggtgac tccatagaag gttgagcgcg 5641 taatagtggg ttggtcttgt ctctatccat tcatctgtga gacatactga tttcaacttt 5701 ttcatcagat aataaggatg gtgtgagata agcaaggctg ggttatgagg tttgggacta 5761 gatggtgttt cgcgtctaaa ggtgagtctt cgttggttca atcacattga tctgatcgct 5821 gatcaattgt cagcctctga atgtgctctt gaagaaaatg gttgtctgta agaatccaga 5881 accaaaaacg gtcatttcaa gtatgtatcc tccgtgaagt tcctttagct cttgctaata 5941 ttggacttta gcgtgcttta gccaaagaca gggtcgcgag gttaagagga tgggccagga 6001 aaaatctaag ttagatttgt cacacaaaga aaaagattgt ctcagttttg gttttgtttg 6061 gtttgattta tgcataggaa atatttatga tcgaaatcac tcatttcctg cttcgtaaat 6121 atcatctact atatgcttcg taaatgtcca tctcctacat attgtggata tgataaatgc 6181 aatcctctcg tctacactct cggcttatgc ctttgtcttc tttgcgctcc ttttagcctt 6241 ctttttattc ctagtcttca agcccatcgg gcgtccagtg cttcgcccgt taccccgacc 6301 acatgaagca gcacgacttc ttcaagtccg ccatgcccga gggctacgtc caggagcgta 6361 ccatcttctt caaggacgac ggcaactaca agacccgtgc cgaggtcaag ttcgagggcg 6421 acaccctcgt caaccgtatc gagctcaagg gcatcgactt caaggaggac ggcaacatcc 6481 tcggccacaa gctcgagtac aactacttct ccgacaacgt ctacatcacc gccgacaagc 6541 agaagaacgg catcaaggcc aacttcaaga tccgtcacaa catcgaggac ggcggcgtcc 6601 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtc ctcctccccg 6661 acaaccacta cctctccacc cagtccaagc tctccaagga ccccaacgag aagcgtgacc 6721 acatggtcct cctcgagttc gtcaccgccg ccggcatcac tctcggcatg gacgagctct 6781 acaagaattc gatatcaagc ttatcgatac cgtcgacctc gagggggggc ccggtaccca 6841 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg 6901 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 6961 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 7021 atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 7081 aatcagctca tttttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag 7141 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga 7201 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg 7261 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc 7321 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg 7381 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc 7441 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca g //