LOCUS       sequence-74369-e        8523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of C-terminal 3XHA 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 8523)
  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 8523)
  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-8XDSR
FEATURES             Location/Qualifiers
     source          1..8523
                     /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(4261..4278)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4261..4277)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4270..4292)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7172..8266
                     /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 ttaaacggca ggttaaacgt aatgttaaac aggtgcttaa
     4201 actttatgtt aaaccccggg ataacttcgt atatggtatt atatacgaag ttatgaattc
     4261 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     4321 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     4381 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     4441 tacgcatctg tgcggtattt cacaccgcat accatcgtcc ggatcaatga agtagatgag
     4501 tttactacct gtatatactt ttttttttgt caaagtcact tttatggcat ttccatgttg
     4561 aaggattagc cacagcacta aattttcagc gtatgattgc ttttaaatat ttaattttca
     4621 tcgtttttta attaatattc aaaacgattt aatgccttct acatctgaag tcaagctcat
     4681 agactcgggt tgggtagcta aaactacatc taacacgaag gggtatagcc ttcttaaagt
     4741 atactcatgt ataaacgttt tgggttcata tttttgggcc acgtggttta taaaaattct
     4801 taactacacc actcggtatc ccgcacccgt ctacgtttct acgatttcga gctaatattg
     4861 agtaaagtga ttagcaaaaa aaataaaagt actcgttgtc ggagatcaag aatttttcta
     4921 ttatctcatc taaaccactt tctaaaagcg aaaaacaaaa tcgtaatatg cagcttgaat
     4981 gggcttccat agtttgaaag aaaaacccta gcagtactgg caagggagac attcctttta
     5041 cctggctaat cgccatcttc cagcaggcgc accattgccc ctgtttcact atccaggtta
     5101 cggatatagt tcatgacaat atttacattg gtccagccac cagcttgcat gatctccggt
     5161 attgaaactc cagcgcgggc catatctcgc gcggctccga cacgggcact gtgtccagac
     5221 caggccaggt atctctgacc agagtcatcc ttagcgccgt aaatcaatcg atgagttgct
     5281 tcaaaaatcc cttccagggc gcgagttgat agctggctgg tggcagatgg cgcggcaaca
     5341 ccattttttc tgacccggca aaacaggtag ttattcggat catcagctac accagagacg
     5401 gaaatccatc gctcgaccag tttagttacc cccaggctaa gtgccttctc tacacctgcg
     5461 gtgctaacca gcgttttcgt tctgccaata tggattaaca ttctcccacc gtcagtacgt
     5521 gagatatctt taaccctgat cctggcaatt tcggctatac gtaacagggt gttataagca
     5581 atccccagaa atgccagatt acgtatatcc tggcagcgat cgctattttc catgagtgaa
     5641 cgaacctggt cgaaatcagt gcgttcgaac gctagagcct gttttgcacg ttcaccggca
     5701 tcaacgtttt cttttcggat ccgccgcata accagtgaaa cagcattgct gtcacttggt
     5761 cgtggcagcc cggaccgacg atgaagcatg tttagctggc ccaaatgttg ctggatagtt
     5821 tttactgcca gaccgcgcgc ctgaagatat agaagataat cgcgaacatc ttcaggttct
     5881 gcgggaaacc atttccggtt attcaacttg caccatgccg cccacgaccg gcaaacggac
     5941 agaagcattt tccaggtatg ctcagaaaac gcctggcgat ccctgaacat gtccatcagg
     6001 ttcttgcgaa cctcatcact cgttgcatcg accggtaact tttagattaa catttgttta
     6061 aatatgaaat taggcaatgt aacatactgc aggcaaattt tggtgtacgg taagtaaatt
     6121 ggacataaga tttaacaaag cgactataag tcagaaagtg agaatgagat tgaaataatt
     6181 aattcaactt attcaattga tgatatgcca ggattcctct tcctttatct tcaatgtttc
     6241 gattaacagg tccattatcc ggtttttagt ttcgctgcac attgccgaat gacaatcgga
     6301 ttcctttaag ctatgctttt gccctgtttt ctctccccag taatcgttgg gtgactgaac
     6361 catccagtag ccgagagtta cgtttttcct gatttaataa atatccatat ctatttgtct
     6421 cgtggtgttt tacagcaact aaccgaaatt actattttgc atcatccaac cataaagaga
     6481 caacaataat tctagaaaac atataacaag tgtttttttc tatttacgtc tcatacgcaa
     6541 agtccacttt accatgtttt ggaaacttgt acccaaccat tcatttttcg attttgtaat
     6601 gaggctcttt tcttctaaca atcattgagg tcattatcat gtgtcctcac ctattgttta
     6661 aattgaggct tgcgctattc ccaaaattgt gggtattatt ttcatatatc agtatctgca
     6721 atttcggttc aactccggaa gctcggtacc caactgtggg aatactcagg tatcgtaaga
     6781 tgcaagagtt cgaatctctt agcaaccatt attttttttc tctcaacata acgagaacac
     6841 acaggggcgc tatcgcacag aatcaaattc gatgactgga aattttttgt taatttcaga
     6901 ggtcgcctga cgcatatacc tttttcaact gaaaaattgg gagaaaaagg aaaggtgaga
     6961 gcgccggaac cggcttttca tatagaatag agaagcgttc atgactaaat gcttgcatca
     7021 caatacttga agttgacaat attatttaag gacctattgt tttttccaat aggtggttag
     7081 caatcgtctt actttctaac ttttcttacc ttttacattt cagcaatata tatatatata
     7141 tatatatata tttcaaggat ataccattgt aatgtctgcc cctaagaaga tcgtcgtttt
     7201 gccaggtgac cacgttggtc aagaaatcac agccgaagcc attaaggttc ttaaagctat
     7261 ttctgatgtt cgttccaatg tcaagttcga tttcgaaaat catttaattg gtggtgctgc
     7321 tatcgatgct acaggtgttc cacttccaga tgaggcgctg gaagcctcca agaaggctga
     7381 tgccgttttg ttaggtgctg tgggtggtcc taaatggggt accggtagtg ttagacctga
     7441 acaaggttta ctaaaaatcc gtaaagaact tcaattgtac gccaacttaa gaccatgtaa
     7501 ctttgcatcc gactctcttt tagacttatc tccaatcaag ccacaatttg ctaaaggtac
     7561 tgacttcgtt gttgttagag aattagtggg aggtatttac tttggtaaga gaaaggaaga
     7621 cgatggtgat ggtgtcgctt gggatagtga acaatacacc gttccagaag tgcaaagaat
     7681 cacaagaatg gccgctttca tggccctaca acatgagcca ccattgccta tttggtcctt
     7741 ggataaagct aatgttttgg cctcttcaag attatggaga aaaactgtgg aggaaaccat
     7801 caagaacgaa tttcctacat tgaaagttca acatcaattg attgattctg ccgccatgat
     7861 cctagttaag aacccaaccc acctaaatgg tattataatc accagcaaca tgtttggtga
     7921 tatcatctcc gatgaagcct ccgttatccc aggctccttg ggtttgttgc catctgcgtc
     7981 cttggcctct ttgccagaca agaacaccgc atttggtttg tacgaaccat gccacggttc
     8041 cgctccagat ttgccaaaga ataaggtcaa ccctatcgcc actatcttgt ctgctgcaat
     8101 gatgttgaaa ttgtcattga acttgcctga agaaggtaaa gccattgaag atgcagttaa
     8161 aaaggttttg gatgcaggta tcagaactgg tgatttaggt ggttccaaca gtaccaccga
     8221 agtcggtgat gctgtcgccg aagaagttaa gaaaatcctt gcttaaaaag attctctttt
     8281 tttgtgatat ttgtacataa actttataaa tgaaattcat aatagaaacg acacgaaatt
     8341 acaaaatgga atatgttcat agggtagacg aaactatata cgcaatctac atacatttat
     8401 caagaaggag aaaaaggagg atgtaaagga atacaggtaa gcaaattgat actaatggct
     8461 caacgtgata aggaaaaaga attgcacttt aacattaata ttgacaagga ggagggcatc
     8521 aca
//
