LOCUS       sequence-71775-e        9057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of C-terminal yEGFP tagged gene 
            sequences into the S. pombe urg1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9057)
  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 9057)
  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
FEATURES             Location/Qualifiers
     source          1..9057
                     /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(4795..4812)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4795..4811)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4804..4826)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7706..8800
                     /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 aaagatctcc cgggataact tcgtatatgg tattatatac gaagttatga attcactggc
     4801 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     4861 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     4921 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca
     4981 tctgtgcggt atttcacacc gcataccatc gtccggatca atgaagtaga tgagtttact
     5041 acctgtatat actttttttt ttgtcaaagt cacttttatg gcatttccat gttgaaggat
     5101 tagccacagc actaaatttt cagcgtatga ttgcttttaa atatttaatt ttcatcgttt
     5161 tttaattaat attcaaaacg atttaatgcc ttctacatct gaagtcaagc tcatagactc
     5221 gggttgggta gctaaaacta catctaacac gaaggggtat agccttctta aagtatactc
     5281 atgtataaac gttttgggtt catatttttg ggccacgtgg tttataaaaa ttcttaacta
     5341 caccactcgg tatcccgcac ccgtctacgt ttctacgatt tcgagctaat attgagtaaa
     5401 gtgattagca aaaaaaataa aagtactcgt tgtcggagat caagaatttt tctattatct
     5461 catctaaacc actttctaaa agcgaaaaac aaaatcgtaa tatgcagctt gaatgggctt
     5521 ccatagtttg aaagaaaaac cctagcagta ctggcaaggg agacattcct tttacctggc
     5581 taatcgccat cttccagcag gcgcaccatt gcccctgttt cactatccag gttacggata
     5641 tagttcatga caatatttac attggtccag ccaccagctt gcatgatctc cggtattgaa
     5701 actccagcgc gggccatatc tcgcgcggct ccgacacggg cactgtgtcc agaccaggcc
     5761 aggtatctct gaccagagtc atccttagcg ccgtaaatca atcgatgagt tgcttcaaaa
     5821 atcccttcca gggcgcgagt tgatagctgg ctggtggcag atggcgcggc aacaccattt
     5881 tttctgaccc ggcaaaacag gtagttattc ggatcatcag ctacaccaga gacggaaatc
     5941 catcgctcga ccagtttagt tacccccagg ctaagtgcct tctctacacc tgcggtgcta
     6001 accagcgttt tcgttctgcc aatatggatt aacattctcc caccgtcagt acgtgagata
     6061 tctttaaccc tgatcctggc aatttcggct atacgtaaca gggtgttata agcaatcccc
     6121 agaaatgcca gattacgtat atcctggcag cgatcgctat tttccatgag tgaacgaacc
     6181 tggtcgaaat cagtgcgttc gaacgctaga gcctgttttg cacgttcacc ggcatcaacg
     6241 ttttcttttc ggatccgccg cataaccagt gaaacagcat tgctgtcact tggtcgtggc
     6301 agcccggacc gacgatgaag catgtttagc tggcccaaat gttgctggat agtttttact
     6361 gccagaccgc gcgcctgaag atatagaaga taatcgcgaa catcttcagg ttctgcggga
     6421 aaccatttcc ggttattcaa cttgcaccat gccgcccacg accggcaaac ggacagaagc
     6481 attttccagg tatgctcaga aaacgcctgg cgatccctga acatgtccat caggttcttg
     6541 cgaacctcat cactcgttgc atcgaccggt aacttttaga ttaacatttg tttaaatatg
     6601 aaattaggca atgtaacata ctgcaggcaa attttggtgt acggtaagta aattggacat
     6661 aagatttaac aaagcgacta taagtcagaa agtgagaatg agattgaaat aattaattca
     6721 acttattcaa ttgatgatat gccaggattc ctcttccttt atcttcaatg tttcgattaa
     6781 caggtccatt atccggtttt tagtttcgct gcacattgcc gaatgacaat cggattcctt
     6841 taagctatgc ttttgccctg ttttctctcc ccagtaatcg ttgggtgact gaaccatcca
     6901 gtagccgaga gttacgtttt tcctgattta ataaatatcc atatctattt gtctcgtggt
     6961 gttttacagc aactaaccga aattactatt ttgcatcatc caaccataaa gagacaacaa
     7021 taattctaga aaacatataa caagtgtttt tttctattta cgtctcatac gcaaagtcca
     7081 ctttaccatg ttttggaaac ttgtacccaa ccattcattt ttcgattttg taatgaggct
     7141 cttttcttct aacaatcatt gaggtcatta tcatgtgtcc tcacctattg tttaaattga
     7201 ggcttgcgct attcccaaaa ttgtgggtat tattttcata tatcagtatc tgcaatttcg
     7261 gttcaactcc ggaagctcgg tacccaactg tgggaatact caggtatcgt aagatgcaag
     7321 agttcgaatc tcttagcaac cattattttt tttctctcaa cataacgaga acacacaggg
     7381 gcgctatcgc acagaatcaa attcgatgac tggaaatttt ttgttaattt cagaggtcgc
     7441 ctgacgcata tacctttttc aactgaaaaa ttgggagaaa aaggaaaggt gagagcgccg
     7501 gaaccggctt ttcatataga atagagaagc gttcatgact aaatgcttgc atcacaatac
     7561 ttgaagttga caatattatt taaggaccta ttgttttttc caataggtgg ttagcaatcg
     7621 tcttactttc taacttttct taccttttac atttcagcaa tatatatata tatatatata
     7681 tatatttcaa ggatatacca ttgtaatgtc tgcccctaag aagatcgtcg ttttgccagg
     7741 tgaccacgtt ggtcaagaaa tcacagccga agccattaag gttcttaaag ctatttctga
     7801 tgttcgttcc aatgtcaagt tcgatttcga aaatcattta attggtggtg ctgctatcga
     7861 tgctacaggt gttccacttc cagatgaggc gctggaagcc tccaagaagg ctgatgccgt
     7921 tttgttaggt gctgtgggtg gtcctaaatg gggtaccggt agtgttagac ctgaacaagg
     7981 tttactaaaa atccgtaaag aacttcaatt gtacgccaac ttaagaccat gtaactttgc
     8041 atccgactct cttttagact tatctccaat caagccacaa tttgctaaag gtactgactt
     8101 cgttgttgtt agagaattag tgggaggtat ttactttggt aagagaaagg aagacgatgg
     8161 tgatggtgtc gcttgggata gtgaacaata caccgttcca gaagtgcaaa gaatcacaag
     8221 aatggccgct ttcatggccc tacaacatga gccaccattg cctatttggt ccttggataa
     8281 agctaatgtt ttggcctctt caagattatg gagaaaaact gtggaggaaa ccatcaagaa
     8341 cgaatttcct acattgaaag ttcaacatca attgattgat tctgccgcca tgatcctagt
     8401 taagaaccca acccacctaa atggtattat aatcaccagc aacatgtttg gtgatatcat
     8461 ctccgatgaa gcctccgtta tcccaggctc cttgggtttg ttgccatctg cgtccttggc
     8521 ctctttgcca gacaagaaca ccgcatttgg tttgtacgaa ccatgccacg gttccgctcc
     8581 agatttgcca aagaataagg tcaaccctat cgccactatc ttgtctgctg caatgatgtt
     8641 gaaattgtca ttgaacttgc ctgaagaagg taaagccatt gaagatgcag ttaaaaaggt
     8701 tttggatgca ggtatcagaa ctggtgattt aggtggttcc aacagtacca ccgaagtcgg
     8761 tgatgctgtc gccgaagaag ttaagaaaat ccttgcttaa aaagattctc tttttttgtg
     8821 atatttgtac ataaacttta taaatgaaat tcataataga aacgacacga aattacaaaa
     8881 tggaatatgt tcatagggta gacgaaacta tatacgcaat ctacatacat ttatcaagaa
     8941 ggagaaaaag gaggatgtaa aggaatacag gtaagcaaat tgatactaat ggctcaacgt
     9001 gataaggaaa aagaattgca ctttaacatt aatattgaca aggaggaggg catcaca
//
