LOCUS       sequence-74364-e        9147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of C-terminal yEGFP tagged gene 
            sequences together with 8 copies of the core DSR motif into the S. 
            pombe urg1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9147)
  AUTHORS   Watson AT, Daigaku Y, Mohebi S, Etheridge TJ, Chahwan C, Murray JM, 
            Carr AM
  TITLE     Optimisation of the Schizosaccharomyces pombe urg1 Expression 
            System.
  JOURNAL   PLoS One. 2013 Dec 20;8(12):e83800. doi: 
            10.1371/journal.pone.0083800.
  PUBMED    24376751
REFERENCE   2  (bases 1 to 9147)
  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: "PLoS One.";
            date: "2013-12-20"; volume: "8(12):e83800. doi"; pages: " 
            10.1371/journal.pone.0083800"
COMMENT     Sequence Label: pAW8ENdeI-cyEGFP-8XDSR
FEATURES             Location/Qualifiers
     source          1..9147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      575..1354
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(1449..1468)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1568..1590
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1628..1646)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1714..1818
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1819..1887,1888..2679)
                     /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(2037..2056)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2850..3438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3339..3358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3592..3609
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3726..3747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3762..3767,3768..3785,3786..3792)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3800..3816
                     /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     3805..3827
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3824..3840
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3824..3840
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(3859..3892)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4026..4742
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             4026..4739
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4885..4902)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4885..4901)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4894..4916)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7796..8890
                     /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 ggtgtagatt 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 atgtttatga
     1201 gtatacctag tctagaaagg cttgtattta aaatattatt caataaaaat tcacaatttt
     1261 tacgacatgt gctaccattc acttaacttc ctgattataa aattggttcg tttatactaa
     1321 ttacttaagt acctttaact aaacaaaatg cctatatata tattaattta caatgagtgt
     1381 cagataagtc actatgtccg agtggttaag gagttagact cgccatcgat ggtatggtgc
     1441 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     1501 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     1561 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     1621 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     1681 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1741 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1801 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1861 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1921 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1981 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2041 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2101 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2161 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2221 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2281 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2341 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     2401 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2461 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2521 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2581 cgtatcgtag ttatctacac gacggggagt caggccacta tggatgaacg aaatagacag
     2641 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2701 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2761 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2821 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2881 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2941 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3001 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3061 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3121 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3181 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3241 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3301 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3361 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3421 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3481 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3541 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3601 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     3661 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3721 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     3781 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     3841 catgattacg ccaagcttat aacttcgtat agcatacatt atacgaagtt attgctagcc
     3901 gttttcttca attactaaat tagaactaat tcaatcatat gctggcatgc ggagagctcg
     3961 gagtcgacga gactagtact ggttctacag gatcaaccgg ctccacgggg tcgatgggta
     4021 gatctatgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg gttgaattag
     4081 atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt gatgctactt
     4141 acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt ccatggccaa
     4201 ccttagtcac tactttaact tatggtgttc aatgtttttc tagataccca gatcacatga
     4261 aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa agaactattt
     4321 ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa ggtgatacct
     4381 tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac attttaggtc
     4441 acaaattgga atacaactat aactctcaca atgtttacat catggctgac aaacaaaaga
     4501 atggtatcaa agttaacttc aaaattagac acaacattga agatggttct gttcaattag
     4561 ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta ccagacaacc
     4621 attacttatc cactcaatct gccttatcca aagatccaaa cgaaaagaga gaccacatgg
     4681 tcttgttaga atttgttact gctgctggta ttacccatgg tatggatgaa ttgtacaaat
     4741 aaagatcttt aaactccgta ttaaacccat tcttaaacag aactttaaac ggcaggttaa
     4801 acgtaatgtt aaacaggtgc ttaaacttta tgttaaaccc cgggataact tcgtatatgg
     4861 tattatatac gaagttatga attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     4921 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     4981 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     5041 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcataccatc
     5101 gtccggatca atgaagtaga tgagtttact acctgtatat actttttttt ttgtcaaagt
     5161 cacttttatg gcatttccat gttgaaggat tagccacagc actaaatttt cagcgtatga
     5221 ttgcttttaa atatttaatt ttcatcgttt tttaattaat attcaaaacg atttaatgcc
     5281 ttctacatct gaagtcaagc tcatagactc gggttgggta gctaaaacta catctaacac
     5341 gaaggggtat agccttctta aagtatactc atgtataaac gttttgggtt catatttttg
     5401 ggccacgtgg tttataaaaa ttcttaacta caccactcgg tatcccgcac ccgtctacgt
     5461 ttctacgatt tcgagctaat attgagtaaa gtgattagca aaaaaaataa aagtactcgt
     5521 tgtcggagat caagaatttt tctattatct catctaaacc actttctaaa agcgaaaaac
     5581 aaaatcgtaa tatgcagctt gaatgggctt ccatagtttg aaagaaaaac cctagcagta
     5641 ctggcaaggg agacattcct tttacctggc taatcgccat cttccagcag gcgcaccatt
     5701 gcccctgttt cactatccag gttacggata tagttcatga caatatttac attggtccag
     5761 ccaccagctt gcatgatctc cggtattgaa actccagcgc gggccatatc tcgcgcggct
     5821 ccgacacggg cactgtgtcc agaccaggcc aggtatctct gaccagagtc atccttagcg
     5881 ccgtaaatca atcgatgagt tgcttcaaaa atcccttcca gggcgcgagt tgatagctgg
     5941 ctggtggcag atggcgcggc aacaccattt tttctgaccc ggcaaaacag gtagttattc
     6001 ggatcatcag ctacaccaga gacggaaatc catcgctcga ccagtttagt tacccccagg
     6061 ctaagtgcct tctctacacc tgcggtgcta accagcgttt tcgttctgcc aatatggatt
     6121 aacattctcc caccgtcagt acgtgagata tctttaaccc tgatcctggc aatttcggct
     6181 atacgtaaca gggtgttata agcaatcccc agaaatgcca gattacgtat atcctggcag
     6241 cgatcgctat tttccatgag tgaacgaacc tggtcgaaat cagtgcgttc gaacgctaga
     6301 gcctgttttg cacgttcacc ggcatcaacg ttttcttttc ggatccgccg cataaccagt
     6361 gaaacagcat tgctgtcact tggtcgtggc agcccggacc gacgatgaag catgtttagc
     6421 tggcccaaat gttgctggat agtttttact gccagaccgc gcgcctgaag atatagaaga
     6481 taatcgcgaa catcttcagg ttctgcggga aaccatttcc ggttattcaa cttgcaccat
     6541 gccgcccacg accggcaaac ggacagaagc attttccagg tatgctcaga aaacgcctgg
     6601 cgatccctga acatgtccat caggttcttg cgaacctcat cactcgttgc atcgaccggt
     6661 aacttttaga ttaacatttg tttaaatatg aaattaggca atgtaacata ctgcaggcaa
     6721 attttggtgt acggtaagta aattggacat aagatttaac aaagcgacta taagtcagaa
     6781 agtgagaatg agattgaaat aattaattca acttattcaa ttgatgatat gccaggattc
     6841 ctcttccttt atcttcaatg tttcgattaa caggtccatt atccggtttt tagtttcgct
     6901 gcacattgcc gaatgacaat cggattcctt taagctatgc ttttgccctg ttttctctcc
     6961 ccagtaatcg ttgggtgact gaaccatcca gtagccgaga gttacgtttt tcctgattta
     7021 ataaatatcc atatctattt gtctcgtggt gttttacagc aactaaccga aattactatt
     7081 ttgcatcatc caaccataaa gagacaacaa taattctaga aaacatataa caagtgtttt
     7141 tttctattta cgtctcatac gcaaagtcca ctttaccatg ttttggaaac ttgtacccaa
     7201 ccattcattt ttcgattttg taatgaggct cttttcttct aacaatcatt gaggtcatta
     7261 tcatgtgtcc tcacctattg tttaaattga ggcttgcgct attcccaaaa ttgtgggtat
     7321 tattttcata tatcagtatc tgcaatttcg gttcaactcc ggaagctcgg tacccaactg
     7381 tgggaatact caggtatcgt aagatgcaag agttcgaatc tcttagcaac cattattttt
     7441 tttctctcaa cataacgaga acacacaggg gcgctatcgc acagaatcaa attcgatgac
     7501 tggaaatttt ttgttaattt cagaggtcgc ctgacgcata tacctttttc aactgaaaaa
     7561 ttgggagaaa aaggaaaggt gagagcgccg gaaccggctt ttcatataga atagagaagc
     7621 gttcatgact aaatgcttgc atcacaatac ttgaagttga caatattatt taaggaccta
     7681 ttgttttttc caataggtgg ttagcaatcg tcttactttc taacttttct taccttttac
     7741 atttcagcaa tatatatata tatatatata tatatttcaa ggatatacca ttgtaatgtc
     7801 tgcccctaag aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa tcacagccga
     7861 agccattaag gttcttaaag ctatttctga tgttcgttcc aatgtcaagt tcgatttcga
     7921 aaatcattta attggtggtg ctgctatcga tgctacaggt gttccacttc cagatgaggc
     7981 gctggaagcc tccaagaagg ctgatgccgt tttgttaggt gctgtgggtg gtcctaaatg
     8041 gggtaccggt agtgttagac ctgaacaagg tttactaaaa atccgtaaag aacttcaatt
     8101 gtacgccaac ttaagaccat gtaactttgc atccgactct cttttagact tatctccaat
     8161 caagccacaa tttgctaaag gtactgactt cgttgttgtt agagaattag tgggaggtat
     8221 ttactttggt aagagaaagg aagacgatgg tgatggtgtc gcttgggata gtgaacaata
     8281 caccgttcca gaagtgcaaa gaatcacaag aatggccgct ttcatggccc tacaacatga
     8341 gccaccattg cctatttggt ccttggataa agctaatgtt ttggcctctt caagattatg
     8401 gagaaaaact gtggaggaaa ccatcaagaa cgaatttcct acattgaaag ttcaacatca
     8461 attgattgat tctgccgcca tgatcctagt taagaaccca acccacctaa atggtattat
     8521 aatcaccagc aacatgtttg gtgatatcat ctccgatgaa gcctccgtta tcccaggctc
     8581 cttgggtttg ttgccatctg cgtccttggc ctctttgcca gacaagaaca ccgcatttgg
     8641 tttgtacgaa ccatgccacg gttccgctcc agatttgcca aagaataagg tcaaccctat
     8701 cgccactatc ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc ctgaagaagg
     8761 taaagccatt gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa ctggtgattt
     8821 aggtggttcc aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag ttaagaaaat
     8881 ccttgcttaa aaagattctc tttttttgtg atatttgtac ataaacttta taaatgaaat
     8941 tcataataga aacgacacga aattacaaaa tggaatatgt tcatagggta gacgaaacta
     9001 tatacgcaat ctacatacat ttatcaagaa ggagaaaaag gaggatgtaa aggaatacag
     9061 gtaagcaaat tgatactaat ggctcaacgt gataaggaaa aagaattgca ctttaacatt
     9121 aatattgaca aggaggaggg catcaca
//
