LOCUS       sequence-74367-e        8475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of C-terminal 3XHA tagged gene 
            sequences together with 4 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 8475)
  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 8475)
  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-c3HA-4XDSR
FEATURES             Location/Qualifiers
     source          1..8475
                     /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..4115
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     primer_bind     complement(4213..4230)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4213..4229)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4222..4244)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7124..8218
                     /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 gatcttaccc atacgatgtt cctgactatg cgggctatcc gtatgacgtc ccggactatg
     4081 caggatccta tccatacgac gttccagatt acgcttaaag atctttaaac tccgtattaa
     4141 acccattctt aaacagaact ttaaaccccg ggataacttc gtatatggta ttatatacga
     4201 agttatgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     4261 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     4321 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     4381 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc ataccatcgt ccggatcaat
     4441 gaagtagatg agtttactac ctgtatatac tttttttttt gtcaaagtca cttttatggc
     4501 atttccatgt tgaaggatta gccacagcac taaattttca gcgtatgatt gcttttaaat
     4561 atttaatttt catcgttttt taattaatat tcaaaacgat ttaatgcctt ctacatctga
     4621 agtcaagctc atagactcgg gttgggtagc taaaactaca tctaacacga aggggtatag
     4681 ccttcttaaa gtatactcat gtataaacgt tttgggttca tatttttggg ccacgtggtt
     4741 tataaaaatt cttaactaca ccactcggta tcccgcaccc gtctacgttt ctacgatttc
     4801 gagctaatat tgagtaaagt gattagcaaa aaaaataaaa gtactcgttg tcggagatca
     4861 agaatttttc tattatctca tctaaaccac tttctaaaag cgaaaaacaa aatcgtaata
     4921 tgcagcttga atgggcttcc atagtttgaa agaaaaaccc tagcagtact ggcaagggag
     4981 acattccttt tacctggcta atcgccatct tccagcaggc gcaccattgc ccctgtttca
     5041 ctatccaggt tacggatata gttcatgaca atatttacat tggtccagcc accagcttgc
     5101 atgatctccg gtattgaaac tccagcgcgg gccatatctc gcgcggctcc gacacgggca
     5161 ctgtgtccag accaggccag gtatctctga ccagagtcat ccttagcgcc gtaaatcaat
     5221 cgatgagttg cttcaaaaat cccttccagg gcgcgagttg atagctggct ggtggcagat
     5281 ggcgcggcaa caccattttt tctgacccgg caaaacaggt agttattcgg atcatcagct
     5341 acaccagaga cggaaatcca tcgctcgacc agtttagtta cccccaggct aagtgccttc
     5401 tctacacctg cggtgctaac cagcgttttc gttctgccaa tatggattaa cattctccca
     5461 ccgtcagtac gtgagatatc tttaaccctg atcctggcaa tttcggctat acgtaacagg
     5521 gtgttataag caatccccag aaatgccaga ttacgtatat cctggcagcg atcgctattt
     5581 tccatgagtg aacgaacctg gtcgaaatca gtgcgttcga acgctagagc ctgttttgca
     5641 cgttcaccgg catcaacgtt ttcttttcgg atccgccgca taaccagtga aacagcattg
     5701 ctgtcacttg gtcgtggcag cccggaccga cgatgaagca tgtttagctg gcccaaatgt
     5761 tgctggatag tttttactgc cagaccgcgc gcctgaagat atagaagata atcgcgaaca
     5821 tcttcaggtt ctgcgggaaa ccatttccgg ttattcaact tgcaccatgc cgcccacgac
     5881 cggcaaacgg acagaagcat tttccaggta tgctcagaaa acgcctggcg atccctgaac
     5941 atgtccatca ggttcttgcg aacctcatca ctcgttgcat cgaccggtaa cttttagatt
     6001 aacatttgtt taaatatgaa attaggcaat gtaacatact gcaggcaaat tttggtgtac
     6061 ggtaagtaaa ttggacataa gatttaacaa agcgactata agtcagaaag tgagaatgag
     6121 attgaaataa ttaattcaac ttattcaatt gatgatatgc caggattcct cttcctttat
     6181 cttcaatgtt tcgattaaca ggtccattat ccggttttta gtttcgctgc acattgccga
     6241 atgacaatcg gattccttta agctatgctt ttgccctgtt ttctctcccc agtaatcgtt
     6301 gggtgactga accatccagt agccgagagt tacgtttttc ctgatttaat aaatatccat
     6361 atctatttgt ctcgtggtgt tttacagcaa ctaaccgaaa ttactatttt gcatcatcca
     6421 accataaaga gacaacaata attctagaaa acatataaca agtgtttttt tctatttacg
     6481 tctcatacgc aaagtccact ttaccatgtt ttggaaactt gtacccaacc attcattttt
     6541 cgattttgta atgaggctct tttcttctaa caatcattga ggtcattatc atgtgtcctc
     6601 acctattgtt taaattgagg cttgcgctat tcccaaaatt gtgggtatta ttttcatata
     6661 tcagtatctg caatttcggt tcaactccgg aagctcggta cccaactgtg ggaatactca
     6721 ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca ttattttttt tctctcaaca
     6781 taacgagaac acacaggggc gctatcgcac agaatcaaat tcgatgactg gaaatttttt
     6841 gttaatttca gaggtcgcct gacgcatata cctttttcaa ctgaaaaatt gggagaaaaa
     6901 ggaaaggtga gagcgccgga accggctttt catatagaat agagaagcgt tcatgactaa
     6961 atgcttgcat cacaatactt gaagttgaca atattattta aggacctatt gttttttcca
     7021 ataggtggtt agcaatcgtc ttactttcta acttttctta ccttttacat ttcagcaata
     7081 tatatatata tatatatata tatttcaagg atataccatt gtaatgtctg cccctaagaa
     7141 gatcgtcgtt ttgccaggtg accacgttgg tcaagaaatc acagccgaag ccattaaggt
     7201 tcttaaagct atttctgatg ttcgttccaa tgtcaagttc gatttcgaaa atcatttaat
     7261 tggtggtgct gctatcgatg ctacaggtgt tccacttcca gatgaggcgc tggaagcctc
     7321 caagaaggct gatgccgttt tgttaggtgc tgtgggtggt cctaaatggg gtaccggtag
     7381 tgttagacct gaacaaggtt tactaaaaat ccgtaaagaa cttcaattgt acgccaactt
     7441 aagaccatgt aactttgcat ccgactctct tttagactta tctccaatca agccacaatt
     7501 tgctaaaggt actgacttcg ttgttgttag agaattagtg ggaggtattt actttggtaa
     7561 gagaaaggaa gacgatggtg atggtgtcgc ttgggatagt gaacaataca ccgttccaga
     7621 agtgcaaaga atcacaagaa tggccgcttt catggcccta caacatgagc caccattgcc
     7681 tatttggtcc ttggataaag ctaatgtttt ggcctcttca agattatgga gaaaaactgt
     7741 ggaggaaacc atcaagaacg aatttcctac attgaaagtt caacatcaat tgattgattc
     7801 tgccgccatg atcctagtta agaacccaac ccacctaaat ggtattataa tcaccagcaa
     7861 catgtttggt gatatcatct ccgatgaagc ctccgttatc ccaggctcct tgggtttgtt
     7921 gccatctgcg tccttggcct ctttgccaga caagaacacc gcatttggtt tgtacgaacc
     7981 atgccacggt tccgctccag atttgccaaa gaataaggtc aaccctatcg ccactatctt
     8041 gtctgctgca atgatgttga aattgtcatt gaacttgcct gaagaaggta aagccattga
     8101 agatgcagtt aaaaaggttt tggatgcagg tatcagaact ggtgatttag gtggttccaa
     8161 cagtaccacc gaagtcggtg atgctgtcgc cgaagaagtt aagaaaatcc ttgcttaaaa
     8221 agattctctt tttttgtgat atttgtacat aaactttata aatgaaattc ataatagaaa
     8281 cgacacgaaa ttacaaaatg gaatatgttc atagggtaga cgaaactata tacgcaatct
     8341 acatacattt atcaagaagg agaaaaagga ggatgtaaag gaatacaggt aagcaaattg
     8401 atactaatgg ctcaacgtga taaggaaaaa gaattgcact ttaacattaa tattgacaag
     8461 gaggagggca tcaca
//
