LOCUS       Exported                6220 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Recycable marker for use in Cryptococcus neoformans. Contains the 
            amdS1 counterselectable marker flanked by direct repeats..
ACCESSION   .
VERSION     .
KEYWORDS    pPEE7
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  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 6220)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6220
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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(1234..2880)
                     /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(4032..4052)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4032..4050)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4071..4087)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4071..4087)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4084..4106)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4095..4111
                     /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(4119..4149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4164..4185
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4302..4319)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4473..5061)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4553..4572)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5232..6092)
                     /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     5855..5874
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6093..6197)
                     /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 aacttggttt atctgtatta acacggaaga gatgtagaaa ctagcttcct ggtttcagag
     1021 acaaggagcc atgaagatcc tgaggattca acggcgttgc tgagggtggg aaggatggta
     1081 ttgttgtctg tagatagatt aatgggcata taatgtcaaa aaagatgatt gacacgatgt
     1141 ctgtctcgaa ttcgcagtgt gctaaaattg tatcacttct ctaatatgta tatatacacc
     1201 ctctaaggaa aattaacccc ttaccgcctt cacctatgga gtcaccacat ttcccagcaa
     1261 cttccccact tcctctgcaa tcgccaacgt cctctcttca ctgagtctcc gtccgataac
     1321 ctgcactgca accggtgccc catggtacgc ctccggatca tactcttcct gcacgagggc
     1381 atcaagctca ctaaccgcct tgaaactctc attcttctta tcgatgttct tatccgcaaa
     1441 ggtaaccgga acaaccacgc tcgtgaaatc cagcaggttg atcacagagg catacccata
     1501 gtaccggaac tggtcatgcc gtaccgcagc ggtaggcgta atcggcgcga tgatggcgtc
     1561 cagttccttc ccggcctttt cttcagcctc ccgccatttc tcaaggtact ccatctggta
     1621 attccacttc tggagatgcg tgtcccagag ctcgttcatg ttaacagctt tgatgttcgg
     1681 gttcagtagg tctttgatat ttggaatcgc cggctcgccg gatgcactga tatcgcgcat
     1741 tacgtcggcg ctgccgtcag ccgcgtagat atgggagatg agatcgtggc cgaaatcgtg
     1801 cttgtatggc gtccacgggg tcacggtgtg accggctttg gcgagtgcgg cgacggtggt
     1861 ttccacgccg cgcaggatag gagggtgtgg aaggacattg ccgtcgaagt tgtagtagcc
     1921 gatattgagc ccgccgttct tgatcttgga ggcaataatg tccgactcgg actggcgcca
     1981 gggcatgggg atgaccttgg agtcgtattt ccatggctcc tgaccgagga cggatttggt
     2041 gaagaggcgg aggtcctcaa cagagtgcgt aatcggcccg acaacgctgt gcaccgtctc
     2101 ctgaccctcc atgctgttcg ccatctttgc atacggcagc cgcccatgac tcggccttag
     2161 accgtacagg aagttgaacg cggccggcac tcgaatcgag ccaccgatat ccgttcctac
     2221 accgatgacg ccaccacgaa tcccaacgat cgcaccctca ccaccagaac tgccgccgca
     2281 cgaccagttc ttgttgcgtg ggttgacggt gcgcccgatg atgttgttga ctgtctcgca
     2341 gaccatcagg gtctgcggga cagaggtctt gacgtagaag acggcaccgg ctttgcggag
     2401 catggttgtc agaaccgagt ccccttcgtc gtacttgttt agccatgaga tgtagcccat
     2461 tgatgtttcg tagcccttga ctcgaagctg gtctttgaga gagatgggga ggccatggag
     2521 tggaccaacg ggtctcttgt gctttgcgta gtattcatcg agttcccttg cctgcgcgag
     2581 agcggcgtca gggaagaact cgtgggcgca gtttgttaac tgctgggcga ttgctgcccg
     2641 tttacagaat gctagcgtaa cttccaccga ggtcaactct ccggccgcca gcttggacac
     2701 aagatctgca gcggaggcct ctgtgatctt cagttcggcc tctgaaagga tccccgattt
     2761 ctttgggaaa tcaataacgc tgtcttccgc aggcagcgtc tggactttcc attcatcagg
     2821 gatggttttt gcgaggcggg cgcgcttatc agcggccagt tcttcccagg attgaggcat
     2881 agacatgttg ggcgagttta ctaatggaaa aagacaatta gcaaagaggc tgcatattag
     2941 gagacgcggt gaagattggg gaggacaata gggtccgtgc accaaaaagt cctgacggca
     3001 gctctgttca acgcacatcc atctcttcca gctatgcgtg tggcaccagc acggatggat
     3061 tgttgcacca aaaggacaag gcgagatgga tgcaggtcga ggcgatgcgg tgctatcggc
     3121 cctcgttgtc gaacgccacg tcgtcctctc ggcgaccgtc aatcctggaa ggaaatgacc
     3181 aagcgtgcac ccagcatcca gcatccaaca tccagcaccc agcacctagc acccatgatc
     3241 caatggccgg catgcagatc agcgtgggga gagcaccatc tagcagcccc tggcaggccc
     3301 agcattgtgc tcggatgtgc gataggcggt gggtgcgata aggtggatta gtggaggatg
     3361 gaggactgag tggcgggcaa gggaaggggg cgggaagatg actcaccttg ggcagtgggt
     3421 gggggatgcg gtatagggga tgtatgtgga tgctggcgga ggatagaagc tgtaagctga
     3481 gctggtgcca acggtgacgc tgtgagagtg gtctcggcga ccctgacggc gactcggcgt
     3541 taccccgtgt ggcacaccgg acgcgcgtcg agtgacgttg ctgctattgt tgttaccatc
     3601 atcctctcct ccgacaacca tacactcagt gcacaccatg ccactcgaat cctgcatgct
     3661 tatgtgagtc ctccccttcc cgttcgtcgt tctccagctc acatcctcgc agctcaatca
     3721 tgccactata ttagaacgag tgcgcagtga gtgagtagag tcaaatggag caaaacgaat
     3781 agtcaacggt aatgaacctg gagtcgtagg ctaatgatta gctgcagtcg gagtcgtaga
     3841 cgcatgagat ccgatagccg gcgacgcatt aggatcagga gtcgaatacg tagatggaag
     3901 ctaactagac gcatgagtat tagtgcgcta atacgtagaa cgagtagtcg gcagttgagg
     3961 attagatggc tagagtagaa cttactagtt ctagagcggc cgccaccgcg gtggagctcc
     4021 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg
     4081 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4141 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4201 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4261 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4321 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4381 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4441 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4501 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4561 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4621 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4681 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4741 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4801 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4861 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4921 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4981 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5041 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5101 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5161 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5221 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5281 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5341 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5401 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5461 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5521 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5581 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5641 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5701 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5761 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5821 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5881 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5941 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6001 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6061 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6121 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6181 caaatagggg ttccgcgcac atttccccga aaagtgccac
//
