LOCUS       sequence-74371-e        9223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of C-terminal HA-auxin-inducible 
            degron tagged gene sequences together with the S. pombe spo5 gene 
            DSR element into the S. pombe urg1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9223)
  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 9223)
  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-cHAIAA7-spo5DSR
FEATURES             Location/Qualifiers
     source          1..9223
                     /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             3981..4007
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4062..4751
                     /codon_start=1
                     /gene="Arabidopsis thaliana AXR3"
                     /product="auxin-responsive IAA17 protein"
                     /label=auxin-inducible degron
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="MMGSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLN
                     NEPANKEGSTTHDVVTFDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSS
                     GGPEAAAFVKVSMDGAPYLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFM
                     NERKLMDLVNSWDYVPSYEDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAM
                     EKCKSRA"
     primer_bind     complement(4961..4978)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4961..4977)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4970..4992)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7872..8966
                     /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 gactagtggt tatccttatg atgttcctga ttatgcttct agtactggtt
     4021 ctacaggatc aaccggctcc acggggtcga tgggtagatc tatgatgggc agtgtcgagc
     4081 tgaatctgag ggagactgag ctgtgtcttg gtcttcccgg tggagataca gtggctccgg
     4141 taaccggaaa caagagaggg ttctcagaga cggttgatct gaagctaaat ctgaataatg
     4201 agcctgcaaa caaggaagga tctacgactc atgacgtcgt gacttttgat tccaaggaga
     4261 agagtgcttg tcctaaagat ccagccaaac ctccggccaa ggcacaagtt gtgggatggc
     4321 caccggtgag atcataccgg aagaacgtga tggtttcctg ccaaaaatca agcggtggcc
     4381 cggaggcggc ggcgttcgtg aaggtatcaa tggacggagc accgtacttg aggaaaatcg
     4441 atttgaggat gtataaaagc tacgatgagc tttctaatgc tttgtccaac atgttcagct
     4501 cttttaccat gggcaaacat ggaggagaag aaggaatgat agacttcatg aatgagagga
     4561 aattgatgga tttggtgaat agctgggact atgttccctc ttatgaagac aaagacggtg
     4621 attggatgct cgtcggcgac gttccttggc caatgttcgt cgatacatgc aagcgtttac
     4681 gtctcatgaa aggatcggat gccattggtc tcgctccgag ggcgatggag aagtgcaaga
     4741 gcagagcttg acccgggact acgccatatc atgcccatcc cgctacttct gctgctaatt
     4801 aaatttaaac atctttccag catttacttg aacatcaaac tctaatatcg aaatcttttc
     4861 aaacacttat cacatactca aacttaaacc aacataaaac ctgttagaca aagccccggg
     4921 ataacttcgt atatggtatt atatacgaag ttatgaattc actggccgtc gttttacaac
     4981 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     5041 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     5101 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     5161 cacaccgcat accatcgtcc ggatcaatga agtagatgag tttactacct gtatatactt
     5221 ttttttttgt caaagtcact tttatggcat ttccatgttg aaggattagc cacagcacta
     5281 aattttcagc gtatgattgc ttttaaatat ttaattttca tcgtttttta attaatattc
     5341 aaaacgattt aatgccttct acatctgaag tcaagctcat agactcgggt tgggtagcta
     5401 aaactacatc taacacgaag gggtatagcc ttcttaaagt atactcatgt ataaacgttt
     5461 tgggttcata tttttgggcc acgtggttta taaaaattct taactacacc actcggtatc
     5521 ccgcacccgt ctacgtttct acgatttcga gctaatattg agtaaagtga ttagcaaaaa
     5581 aaataaaagt actcgttgtc ggagatcaag aatttttcta ttatctcatc taaaccactt
     5641 tctaaaagcg aaaaacaaaa tcgtaatatg cagcttgaat gggcttccat agtttgaaag
     5701 aaaaacccta gcagtactgg caagggagac attcctttta cctggctaat cgccatcttc
     5761 cagcaggcgc accattgccc ctgtttcact atccaggtta cggatatagt tcatgacaat
     5821 atttacattg gtccagccac cagcttgcat gatctccggt attgaaactc cagcgcgggc
     5881 catatctcgc gcggctccga cacgggcact gtgtccagac caggccaggt atctctgacc
     5941 agagtcatcc ttagcgccgt aaatcaatcg atgagttgct tcaaaaatcc cttccagggc
     6001 gcgagttgat agctggctgg tggcagatgg cgcggcaaca ccattttttc tgacccggca
     6061 aaacaggtag ttattcggat catcagctac accagagacg gaaatccatc gctcgaccag
     6121 tttagttacc cccaggctaa gtgccttctc tacacctgcg gtgctaacca gcgttttcgt
     6181 tctgccaata tggattaaca ttctcccacc gtcagtacgt gagatatctt taaccctgat
     6241 cctggcaatt tcggctatac gtaacagggt gttataagca atccccagaa atgccagatt
     6301 acgtatatcc tggcagcgat cgctattttc catgagtgaa cgaacctggt cgaaatcagt
     6361 gcgttcgaac gctagagcct gttttgcacg ttcaccggca tcaacgtttt cttttcggat
     6421 ccgccgcata accagtgaaa cagcattgct gtcacttggt cgtggcagcc cggaccgacg
     6481 atgaagcatg tttagctggc ccaaatgttg ctggatagtt tttactgcca gaccgcgcgc
     6541 ctgaagatat agaagataat cgcgaacatc ttcaggttct gcgggaaacc atttccggtt
     6601 attcaacttg caccatgccg cccacgaccg gcaaacggac agaagcattt tccaggtatg
     6661 ctcagaaaac gcctggcgat ccctgaacat gtccatcagg ttcttgcgaa cctcatcact
     6721 cgttgcatcg accggtaact tttagattaa catttgttta aatatgaaat taggcaatgt
     6781 aacatactgc aggcaaattt tggtgtacgg taagtaaatt ggacataaga tttaacaaag
     6841 cgactataag tcagaaagtg agaatgagat tgaaataatt aattcaactt attcaattga
     6901 tgatatgcca ggattcctct tcctttatct tcaatgtttc gattaacagg tccattatcc
     6961 ggtttttagt ttcgctgcac attgccgaat gacaatcgga ttcctttaag ctatgctttt
     7021 gccctgtttt ctctccccag taatcgttgg gtgactgaac catccagtag ccgagagtta
     7081 cgtttttcct gatttaataa atatccatat ctatttgtct cgtggtgttt tacagcaact
     7141 aaccgaaatt actattttgc atcatccaac cataaagaga caacaataat tctagaaaac
     7201 atataacaag tgtttttttc tatttacgtc tcatacgcaa agtccacttt accatgtttt
     7261 ggaaacttgt acccaaccat tcatttttcg attttgtaat gaggctcttt tcttctaaca
     7321 atcattgagg tcattatcat gtgtcctcac ctattgttta aattgaggct tgcgctattc
     7381 ccaaaattgt gggtattatt ttcatatatc agtatctgca atttcggttc aactccggaa
     7441 gctcggtacc caactgtggg aatactcagg tatcgtaaga tgcaagagtt cgaatctctt
     7501 agcaaccatt attttttttc tctcaacata acgagaacac acaggggcgc tatcgcacag
     7561 aatcaaattc gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc
     7621 tttttcaact gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca
     7681 tatagaatag agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat
     7741 attatttaag gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac
     7801 ttttcttacc ttttacattt cagcaatata tatatatata tatatatata tttcaaggat
     7861 ataccattgt aatgtctgcc cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc
     7921 aagaaatcac agccgaagcc attaaggttc ttaaagctat ttctgatgtt cgttccaatg
     7981 tcaagttcga tttcgaaaat catttaattg gtggtgctgc tatcgatgct acaggtgttc
     8041 cacttccaga tgaggcgctg gaagcctcca agaaggctga tgccgttttg ttaggtgctg
     8101 tgggtggtcc taaatggggt accggtagtg ttagacctga acaaggttta ctaaaaatcc
     8161 gtaaagaact tcaattgtac gccaacttaa gaccatgtaa ctttgcatcc gactctcttt
     8221 tagacttatc tccaatcaag ccacaatttg ctaaaggtac tgacttcgtt gttgttagag
     8281 aattagtggg aggtatttac tttggtaaga gaaaggaaga cgatggtgat ggtgtcgctt
     8341 gggatagtga acaatacacc gttccagaag tgcaaagaat cacaagaatg gccgctttca
     8401 tggccctaca acatgagcca ccattgccta tttggtcctt ggataaagct aatgttttgg
     8461 cctcttcaag attatggaga aaaactgtgg aggaaaccat caagaacgaa tttcctacat
     8521 tgaaagttca acatcaattg attgattctg ccgccatgat cctagttaag aacccaaccc
     8581 acctaaatgg tattataatc accagcaaca tgtttggtga tatcatctcc gatgaagcct
     8641 ccgttatccc aggctccttg ggtttgttgc catctgcgtc cttggcctct ttgccagaca
     8701 agaacaccgc atttggtttg tacgaaccat gccacggttc cgctccagat ttgccaaaga
     8761 ataaggtcaa ccctatcgcc actatcttgt ctgctgcaat gatgttgaaa ttgtcattga
     8821 acttgcctga agaaggtaaa gccattgaag atgcagttaa aaaggttttg gatgcaggta
     8881 tcagaactgg tgatttaggt ggttccaaca gtaccaccga agtcggtgat gctgtcgccg
     8941 aagaagttaa gaaaatcctt gcttaaaaag attctctttt tttgtgatat ttgtacataa
     9001 actttataaa tgaaattcat aatagaaacg acacgaaatt acaaaatgga atatgttcat
     9061 agggtagacg aaactatata cgcaatctac atacatttat caagaaggag aaaaaggagg
     9121 atgtaaagga atacaggtaa gcaaattgat actaatggct caacgtgata aggaaaaaga
     9181 attgcacttt aacattaata ttgacaagga ggagggcatc aca
//
