LOCUS       Exported                8252 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Cre-expression plasmid for cassette exchange.
ACCESSION   .
VERSION     .
KEYWORDS    pAW8
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8252)
  AUTHORS   Watson AT1, Garcia V, Bone N, Carr AM, Armstrong J.
  TITLE     Gene tagging and gene replacement using recombinase-mediated 
            cassette exchange in Schizosaccharomyces pombe.
  JOURNAL   Gene. 2008 Jan 15;407(1-2):63-74
  PUBMED    18054176
REFERENCE   2  (bases 1 to 8252)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8252
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      575..1356
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(1451..1470)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1570..1592
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1630..1648)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1716..1820
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1821..2681
                     /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(2039..2058)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2852..3440
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3341..3360
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3594..3611
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3728..3749
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3764..3794
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3802..3818
                     /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     3807..3829
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3826..3842
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3826..3842
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3838..4224
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHGGRITSYSIHYTKLCMRRARSRRTSITSYMVLYTKL
                     CGRANSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWR
                     LMRYFLLTHLCGISHRIPSSGSMK"
     protein_bind    3874..3907
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(3990..4007)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3990..4006)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3999..4021)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             6901..7995
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
ORIGIN
        1 caaaaagtta ggtgtaacag aaaatcatga aactatgatt tctaatttat atattggagg
       61 attttctcta aaaaaaaaaa tacaacaaat aaaaaacact caatgacctg accatttgat
      121 ggagtttaag tcaatacctt cttgaaccat ttcccataat ggtgaaagtt ccctcaagaa
      181 ttttactctg tcagaaacgg ccttaacgac gtagtcgatc cggacgatgg ccaaccttcc
      241 aattcattaa atcgagtcct ccatcagatg atttacaagg ttttgcatag aatccctaag
      301 taagagaagt tgttagtgat ttcttcaagt ttactaaaat ttacatacaa atggatgatc
      361 tcttctccat tgtttccttt cttcttgcaa acgcgtttta caaagagatg acatgctatt
      421 ttctgattat ttttttctat agttttctat tttgtacgtc cttgggagtc cgaaatgtaa
      481 aatcggggta tggttagtag cggcagcgtt cattatggta tataaaagat aaaaagtagg
      541 ggcaagcgag acagaatggg atacaagggc atcgtctata attatagcta aaaattgtat
      601 tttaatttgt attttttgta attttatttc tcattgtttt acttaaaatg aatgcgaatt
      661 agagaaaact tcaacgaaat gtcaaaataa gctcagcaaa atatacaatt ttagggaaag
      721 cgatcagcaa cttattctcc gttgtagatt ctttttcttc ataaattcca acaatgtaac
      781 tgccaattga tttttctcta gatgctaata aattgaaact aatgaatata ataacacagg
      841 ttttgataaa ttgtaaactc ttactcaaaa aaagaatgtt aaaactaatt taaccttttt
      901 ataaaggctc atggcgttaa agaggatagt aaaaattcta caatttttta atttaatgtc
      961 acaattgtct tgcgaatata attctattaa ttttcgctaa ctgctgtttt agtactatta
     1021 ctttcaatat gccttatatg caactttaat tcatgaagaa tgcaatattt ttgcaatctt
     1081 tcttgtactg agcgcttttt atcatttagt actcattatt taatttttca gtaaagggca
     1141 ataaataaat ttgtagaaga tgcaatgtaa tctcctctat ccttttgctc atatgtttat
     1201 gagtatacct agtctagaaa ggcttgtatt taaaatatta ttcaataaaa attcacaatt
     1261 tttacgacat gtgctaccat tcacttaact tcctgattat aaaattggtt cgtttatact
     1321 aattacttaa gtacctttaa ctaaacaaaa tgcctatata tatattaatt tacaatgagt
     1381 gtcagataag tcactatgtc cgagtggtta aggagttaga ctcgccatcg atggtatggt
     1441 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     1501 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     1561 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     1621 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1681 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1741 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1801 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1861 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1921 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1981 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2041 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     2101 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2161 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2221 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2281 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2341 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     2401 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2461 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2521 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2581 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2641 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2701 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2761 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2821 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2881 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2941 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     3001 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3061 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3121 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3181 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3241 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3301 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3361 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3421 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3481 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3541 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3601 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     3661 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     3721 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     3781 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     3841 accatgatta cgccaagctt gcatggcggc cgcataactt cgtatagcat acattatacg
     3901 aagttatgca tgcggagagc tcggagtcga cgaactagta taacttcgta tatggtatta
     3961 tatacgaagt tatgcggccg cgcgaattca ctggccgtcg ttttacaacg tcgtgactgg
     4021 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     4081 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     4141 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4201 ccatcgtccg gatcaatgaa gtagatgagt ttactacctg tatatacttt tttttttgtc
     4261 aaagtcactt ttatggcatt tccatgttga aggattagcc acagcactaa attttcagcg
     4321 tatgattgct tttaaatatt taattttcat cgttttttaa ttaatattca aaacgattta
     4381 atgccttcta catctgaagt caagctcata gactcgggtt gggtagctaa aactacatct
     4441 aacacgaagg ggtatagcct tcttaaagta tactcatgta taaacgtttt gggttcatat
     4501 ttttgggcca cgtggtttat aaaaattctt aactacacca ctcggtatcc cgcacccgtc
     4561 tacgtttcta cgatttcgag ctaatattga gtaaagtgat tagcaaaaaa aataaaagta
     4621 ctcgttgtcg gagatcaaga atttttctat tatctcatct aaaccacttt ctaaaagcga
     4681 aaaacaaaat cgtaatatgc agcttgaatg ggcttccata gtttgaaaga aaaaccctag
     4741 cagtactggc aagggagaca ttccttttac ctggctaatc gccatcttcc agcaggcgca
     4801 ccattgcccc tgtttcacta tccaggttac ggatatagtt catgacaata tttacattgg
     4861 tccagccacc agcttgcatg atctccggta ttgaaactcc agcgcgggcc atatctcgcg
     4921 cggctccgac acgggcactg tgtccagacc aggccaggta tctctgacca gagtcatcct
     4981 tagcgccgta aatcaatcga tgagttgctt caaaaatccc ttccagggcg cgagttgata
     5041 gctggctggt ggcagatggc gcggcaacac cattttttct gacccggcaa aacaggtagt
     5101 tattcggatc atcagctaca ccagagacgg aaatccatcg ctcgaccagt ttagttaccc
     5161 ccaggctaag tgccttctct acacctgcgg tgctaaccag cgttttcgtt ctgccaatat
     5221 ggattaacat tctcccaccg tcagtacgtg agatatcttt aaccctgatc ctggcaattt
     5281 cggctatacg taacagggtg ttataagcaa tccccagaaa tgccagatta cgtatatcct
     5341 ggcagcgatc gctattttcc atgagtgaac gaacctggtc gaaatcagtg cgttcgaacg
     5401 ctagagcctg ttttgcacgt tcaccggcat caacgttttc ttttcggatc cgccgcataa
     5461 ccagtgaaac agcattgctg tcacttggtc gtggcagccc ggaccgacga tgaagcatgt
     5521 ttagctggcc caaatgttgc tggatagttt ttactgccag accgcgcgcc tgaagatata
     5581 gaagataatc gcgaacatct tcaggttctg cgggaaacca tttccggtta ttcaacttgc
     5641 accatgccgc ccacgaccgg caaacggaca gaagcatttt ccaggtatgc tcagaaaacg
     5701 cctggcgatc cctgaacatg tccatcaggt tcttgcgaac ctcatcactc gttgcatcga
     5761 ccggtaactt ttagattaac atttgtttaa atatgaaatt aggcaatgta acatactgca
     5821 ggcaaatttt ggtgtacggt aagtaaattg gacatatgat ttaacaaagc gactataagt
     5881 cagaaagtga gaatgagatt gaaataatta attcaactta ttcaattgat gatatgccag
     5941 gattcctctt cctttatctt caatgtttcg attaacaggt ccattatccg gtttttagtt
     6001 tcgctgcaca ttgccgaatg acaatcggat tcctttaagc tttgcttttg ccctgttttc
     6061 tctccccagt aatcgttggg tgactgaacc atccagtagc cgagagttac gtttttcctg
     6121 atttaataaa tatccatatc tatttgtctc gtggtgtttt acagcaacta accgaaatta
     6181 ctattttgca tcatccaacc ataaagagac aacaataatt ctagaaaaca tataacaagt
     6241 gtttttttct atttacgtct catacgcaaa gtccacttta ccatgttttg gaaacttgta
     6301 cccaaccatt catttttcga ttttgtaatg aggctctttt cttctaacaa tcattgaggt
     6361 cattatcatg tgtcctcacc tattgtttaa attgaggctt gcgctattcc caaaattgtg
     6421 ggtattattt tcatatatca gtatctgcaa tttcggttca actccggaag ctcggtaccc
     6481 aactgtggga atactcaggt atcgtaagat gcaagagttc gaatctctta gcaaccatta
     6541 ttttttttct ctcaacataa cgagaacaca caggggcgct atcgcacaga atcaaattcg
     6601 atgactggaa attttttgtt aatttcagag gtcgcctgac gcatatacct ttttcaactg
     6661 aaaaattggg agaaaaagga aaggtgagag cgccggaacc ggcttttcat atagaataga
     6721 gaagcgttca tgactaaatg cttgcatcac aatacttgaa gttgacaata ttatttaagg
     6781 acctattgtt ttttccaata ggtggttagc aatcgtctta ctttctaact tttcttacct
     6841 tttacatttc agcaatatat atatatatat atatatatat ttcaaggata taccattgta
     6901 atgtctgccc ctaagaagat cgtcgttttg ccaggtgacc acgttggtca agaaatcaca
     6961 gccgaagcca ttaaggttct taaagctatt tctgatgttc gttccaatgt caagttcgat
     7021 ttcgaaaatc atttaattgg tggtgctgct atcgatgcta caggtgttcc acttccagat
     7081 gaggcgctgg aagcctccaa gaaggctgat gccgttttgt taggtgctgt gggtggtcct
     7141 aaatggggta ccggtagtgt tagacctgaa caaggtttac taaaaatccg taaagaactt
     7201 caattgtacg ccaacttaag accatgtaac tttgcatccg actctctttt agacttatct
     7261 ccaatcaagc cacaatttgc taaaggtact gacttcgttg ttgttagaga attagtggga
     7321 ggtatttact ttggtaagag aaaggaagac gatggtgatg gtgtcgcttg ggatagtgaa
     7381 caatacaccg ttccagaagt gcaaagaatc acaagaatgg ccgctttcat ggccctacaa
     7441 catgagccac cattgcctat ttggtccttg gataaagcta atgttttggc ctcttcaaga
     7501 ttatggagaa aaactgtgga ggaaaccatc aagaacgaat ttcctacatt gaaagttcaa
     7561 catcaattga ttgattctgc cgccatgatc ctagttaaga acccaaccca cctaaatggt
     7621 attataatca ccagcaacat gtttggtgat atcatctccg atgaagcctc cgttatccca
     7681 ggctccttgg gtttgttgcc atctgcgtcc ttggcctctt tgccagacaa gaacaccgca
     7741 tttggtttgt acgaaccatg ccatggttcc gctccagatt tgccaaagaa taaggtcaac
     7801 cctatcgcca ctatcttgtc tgctgcaatg atgttgaaat tgtcattgaa cttgcctgaa
     7861 gaaggtaaag ccattgaaga tgcagttaaa aaggttttgg atgcaggtat cagaactggt
     7921 gatttaggtg gttccaacag taccaccgaa gtcggtgatg ctgtcgccga agaagttaag
     7981 aaaatccttg cttaaaaaga ttctcttttt ttgtgatatt tgtacataaa ctttataaat
     8041 gaaattcata atagaaacga cacgaaatta caaaatggaa tatgttcata gggtagacga
     8101 aactatatac gcaatctaca tacatttatc aagaaggaga aaaaggagga tgtaaaggaa
     8161 tacaggtaag caaattgata ctaatggctc aacgtgataa ggaaaaagaa ttgcacttta
     8221 acattaatat tgacaaggag gagggcatca ca
//
