LOCUS       sequence-71777-e        9060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of C-terminal HA-auxin-inducible 
            degron 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 9060)
  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 9060)
  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-cHAIAA17
FEATURES             Location/Qualifiers
     source          1..9060
                     /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(4798..4815)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4798..4814)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4807..4829)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7709..8803
                     /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 acccgggata acttcgtata tggtattata tacgaagtta tgaattcact
     4801 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4861 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4921 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     4981 gcatctgtgc ggtatttcac accgcatacc atcgtccgga tcaatgaagt agatgagttt
     5041 actacctgta tatacttttt tttttgtcaa agtcactttt atggcatttc catgttgaag
     5101 gattagccac agcactaaat tttcagcgta tgattgcttt taaatattta attttcatcg
     5161 ttttttaatt aatattcaaa acgatttaat gccttctaca tctgaagtca agctcataga
     5221 ctcgggttgg gtagctaaaa ctacatctaa cacgaagggg tatagccttc ttaaagtata
     5281 ctcatgtata aacgttttgg gttcatattt ttgggccacg tggtttataa aaattcttaa
     5341 ctacaccact cggtatcccg cacccgtcta cgtttctacg atttcgagct aatattgagt
     5401 aaagtgatta gcaaaaaaaa taaaagtact cgttgtcgga gatcaagaat ttttctatta
     5461 tctcatctaa accactttct aaaagcgaaa aacaaaatcg taatatgcag cttgaatggg
     5521 cttccatagt ttgaaagaaa aaccctagca gtactggcaa gggagacatt ccttttacct
     5581 ggctaatcgc catcttccag caggcgcacc attgcccctg tttcactatc caggttacgg
     5641 atatagttca tgacaatatt tacattggtc cagccaccag cttgcatgat ctccggtatt
     5701 gaaactccag cgcgggccat atctcgcgcg gctccgacac gggcactgtg tccagaccag
     5761 gccaggtatc tctgaccaga gtcatcctta gcgccgtaaa tcaatcgatg agttgcttca
     5821 aaaatccctt ccagggcgcg agttgatagc tggctggtgg cagatggcgc ggcaacacca
     5881 ttttttctga cccggcaaaa caggtagtta ttcggatcat cagctacacc agagacggaa
     5941 atccatcgct cgaccagttt agttaccccc aggctaagtg ccttctctac acctgcggtg
     6001 ctaaccagcg ttttcgttct gccaatatgg attaacattc tcccaccgtc agtacgtgag
     6061 atatctttaa ccctgatcct ggcaatttcg gctatacgta acagggtgtt ataagcaatc
     6121 cccagaaatg ccagattacg tatatcctgg cagcgatcgc tattttccat gagtgaacga
     6181 acctggtcga aatcagtgcg ttcgaacgct agagcctgtt ttgcacgttc accggcatca
     6241 acgttttctt ttcggatccg ccgcataacc agtgaaacag cattgctgtc acttggtcgt
     6301 ggcagcccgg accgacgatg aagcatgttt agctggccca aatgttgctg gatagttttt
     6361 actgccagac cgcgcgcctg aagatataga agataatcgc gaacatcttc aggttctgcg
     6421 ggaaaccatt tccggttatt caacttgcac catgccgccc acgaccggca aacggacaga
     6481 agcattttcc aggtatgctc agaaaacgcc tggcgatccc tgaacatgtc catcaggttc
     6541 ttgcgaacct catcactcgt tgcatcgacc ggtaactttt agattaacat ttgtttaaat
     6601 atgaaattag gcaatgtaac atactgcagg caaattttgg tgtacggtaa gtaaattgga
     6661 cataagattt aacaaagcga ctataagtca gaaagtgaga atgagattga aataattaat
     6721 tcaacttatt caattgatga tatgccagga ttcctcttcc tttatcttca atgtttcgat
     6781 taacaggtcc attatccggt ttttagtttc gctgcacatt gccgaatgac aatcggattc
     6841 ctttaagcta tgcttttgcc ctgttttctc tccccagtaa tcgttgggtg actgaaccat
     6901 ccagtagccg agagttacgt ttttcctgat ttaataaata tccatatcta tttgtctcgt
     6961 ggtgttttac agcaactaac cgaaattact attttgcatc atccaaccat aaagagacaa
     7021 caataattct agaaaacata taacaagtgt ttttttctat ttacgtctca tacgcaaagt
     7081 ccactttacc atgttttgga aacttgtacc caaccattca tttttcgatt ttgtaatgag
     7141 gctcttttct tctaacaatc attgaggtca ttatcatgtg tcctcaccta ttgtttaaat
     7201 tgaggcttgc gctattccca aaattgtggg tattattttc atatatcagt atctgcaatt
     7261 tcggttcaac tccggaagct cggtacccaa ctgtgggaat actcaggtat cgtaagatgc
     7321 aagagttcga atctcttagc aaccattatt ttttttctct caacataacg agaacacaca
     7381 ggggcgctat cgcacagaat caaattcgat gactggaaat tttttgttaa tttcagaggt
     7441 cgcctgacgc atataccttt ttcaactgaa aaattgggag aaaaaggaaa ggtgagagcg
     7501 ccggaaccgg cttttcatat agaatagaga agcgttcatg actaaatgct tgcatcacaa
     7561 tacttgaagt tgacaatatt atttaaggac ctattgtttt ttccaatagg tggttagcaa
     7621 tcgtcttact ttctaacttt tcttaccttt tacatttcag caatatatat atatatatat
     7681 atatatattt caaggatata ccattgtaat gtctgcccct aagaagatcg tcgttttgcc
     7741 aggtgaccac gttggtcaag aaatcacagc cgaagccatt aaggttctta aagctatttc
     7801 tgatgttcgt tccaatgtca agttcgattt cgaaaatcat ttaattggtg gtgctgctat
     7861 cgatgctaca ggtgttccac ttccagatga ggcgctggaa gcctccaaga aggctgatgc
     7921 cgttttgtta ggtgctgtgg gtggtcctaa atggggtacc ggtagtgtta gacctgaaca
     7981 aggtttacta aaaatccgta aagaacttca attgtacgcc aacttaagac catgtaactt
     8041 tgcatccgac tctcttttag acttatctcc aatcaagcca caatttgcta aaggtactga
     8101 cttcgttgtt gttagagaat tagtgggagg tatttacttt ggtaagagaa aggaagacga
     8161 tggtgatggt gtcgcttggg atagtgaaca atacaccgtt ccagaagtgc aaagaatcac
     8221 aagaatggcc gctttcatgg ccctacaaca tgagccacca ttgcctattt ggtccttgga
     8281 taaagctaat gttttggcct cttcaagatt atggagaaaa actgtggagg aaaccatcaa
     8341 gaacgaattt cctacattga aagttcaaca tcaattgatt gattctgccg ccatgatcct
     8401 agttaagaac ccaacccacc taaatggtat tataatcacc agcaacatgt ttggtgatat
     8461 catctccgat gaagcctccg ttatcccagg ctccttgggt ttgttgccat ctgcgtcctt
     8521 ggcctctttg ccagacaaga acaccgcatt tggtttgtac gaaccatgcc acggttccgc
     8581 tccagatttg ccaaagaata aggtcaaccc tatcgccact atcttgtctg ctgcaatgat
     8641 gttgaaattg tcattgaact tgcctgaaga aggtaaagcc attgaagatg cagttaaaaa
     8701 ggttttggat gcaggtatca gaactggtga tttaggtggt tccaacagta ccaccgaagt
     8761 cggtgatgct gtcgccgaag aagttaagaa aatccttgct taaaaagatt ctcttttttt
     8821 gtgatatttg tacataaact ttataaatga aattcataat agaaacgaca cgaaattaca
     8881 aaatggaata tgttcatagg gtagacgaaa ctatatacgc aatctacata catttatcaa
     8941 gaaggagaaa aaggaggatg taaaggaata caggtaagca aattgatact aatggctcaa
     9001 cgtgataagg aaaaagaatt gcactttaac attaatattg acaaggagga gggcatcaca
//
