LOCUS       sequence-74370-e        8596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of C-terminal 3XHA 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 8596)
  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 8596)
  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-spo5DSR
FEATURES             Location/Qualifiers
     source          1..8596
                     /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(4334..4351)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4334..4350)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4343..4365)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7245..8339
                     /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 atctcccggg actacgccat
     4141 atcatgccca tcccgctact tctgctgcta attaaattta aacatctttc cagcatttac
     4201 ttgaacatca aactctaata tcgaaatctt ttcaaacact tatcacatac tcaaacttaa
     4261 accaacataa aacctgttag acaaagcccc gggataactt cgtatatggt attatatacg
     4321 aagttatgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4381 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4441 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     4501 ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataccatcg tccggatcaa
     4561 tgaagtagat gagtttacta cctgtatata cttttttttt tgtcaaagtc acttttatgg
     4621 catttccatg ttgaaggatt agccacagca ctaaattttc agcgtatgat tgcttttaaa
     4681 tatttaattt tcatcgtttt ttaattaata ttcaaaacga tttaatgcct tctacatctg
     4741 aagtcaagct catagactcg ggttgggtag ctaaaactac atctaacacg aaggggtata
     4801 gccttcttaa agtatactca tgtataaacg ttttgggttc atatttttgg gccacgtggt
     4861 ttataaaaat tcttaactac accactcggt atcccgcacc cgtctacgtt tctacgattt
     4921 cgagctaata ttgagtaaag tgattagcaa aaaaaataaa agtactcgtt gtcggagatc
     4981 aagaattttt ctattatctc atctaaacca ctttctaaaa gcgaaaaaca aaatcgtaat
     5041 atgcagcttg aatgggcttc catagtttga aagaaaaacc ctagcagtac tggcaaggga
     5101 gacattcctt ttacctggct aatcgccatc ttccagcagg cgcaccattg cccctgtttc
     5161 actatccagg ttacggatat agttcatgac aatatttaca ttggtccagc caccagcttg
     5221 catgatctcc ggtattgaaa ctccagcgcg ggccatatct cgcgcggctc cgacacgggc
     5281 actgtgtcca gaccaggcca ggtatctctg accagagtca tccttagcgc cgtaaatcaa
     5341 tcgatgagtt gcttcaaaaa tcccttccag ggcgcgagtt gatagctggc tggtggcaga
     5401 tggcgcggca acaccatttt ttctgacccg gcaaaacagg tagttattcg gatcatcagc
     5461 tacaccagag acggaaatcc atcgctcgac cagtttagtt acccccaggc taagtgcctt
     5521 ctctacacct gcggtgctaa ccagcgtttt cgttctgcca atatggatta acattctccc
     5581 accgtcagta cgtgagatat ctttaaccct gatcctggca atttcggcta tacgtaacag
     5641 ggtgttataa gcaatcccca gaaatgccag attacgtata tcctggcagc gatcgctatt
     5701 ttccatgagt gaacgaacct ggtcgaaatc agtgcgttcg aacgctagag cctgttttgc
     5761 acgttcaccg gcatcaacgt tttcttttcg gatccgccgc ataaccagtg aaacagcatt
     5821 gctgtcactt ggtcgtggca gcccggaccg acgatgaagc atgtttagct ggcccaaatg
     5881 ttgctggata gtttttactg ccagaccgcg cgcctgaaga tatagaagat aatcgcgaac
     5941 atcttcaggt tctgcgggaa accatttccg gttattcaac ttgcaccatg ccgcccacga
     6001 ccggcaaacg gacagaagca ttttccaggt atgctcagaa aacgcctggc gatccctgaa
     6061 catgtccatc aggttcttgc gaacctcatc actcgttgca tcgaccggta acttttagat
     6121 taacatttgt ttaaatatga aattaggcaa tgtaacatac tgcaggcaaa ttttggtgta
     6181 cggtaagtaa attggacata agatttaaca aagcgactat aagtcagaaa gtgagaatga
     6241 gattgaaata attaattcaa cttattcaat tgatgatatg ccaggattcc tcttccttta
     6301 tcttcaatgt ttcgattaac aggtccatta tccggttttt agtttcgctg cacattgccg
     6361 aatgacaatc ggattccttt aagctatgct tttgccctgt tttctctccc cagtaatcgt
     6421 tgggtgactg aaccatccag tagccgagag ttacgttttt cctgatttaa taaatatcca
     6481 tatctatttg tctcgtggtg ttttacagca actaaccgaa attactattt tgcatcatcc
     6541 aaccataaag agacaacaat aattctagaa aacatataac aagtgttttt ttctatttac
     6601 gtctcatacg caaagtccac tttaccatgt tttggaaact tgtacccaac cattcatttt
     6661 tcgattttgt aatgaggctc ttttcttcta acaatcattg aggtcattat catgtgtcct
     6721 cacctattgt ttaaattgag gcttgcgcta ttcccaaaat tgtgggtatt attttcatat
     6781 atcagtatct gcaatttcgg ttcaactccg gaagctcggt acccaactgt gggaatactc
     6841 aggtatcgta agatgcaaga gttcgaatct cttagcaacc attatttttt ttctctcaac
     6901 ataacgagaa cacacagggg cgctatcgca cagaatcaaa ttcgatgact ggaaattttt
     6961 tgttaatttc agaggtcgcc tgacgcatat acctttttca actgaaaaat tgggagaaaa
     7021 aggaaaggtg agagcgccgg aaccggcttt tcatatagaa tagagaagcg ttcatgacta
     7081 aatgcttgca tcacaatact tgaagttgac aatattattt aaggacctat tgttttttcc
     7141 aataggtggt tagcaatcgt cttactttct aacttttctt accttttaca tttcagcaat
     7201 atatatatat atatatatat atatttcaag gatataccat tgtaatgtct gcccctaaga
     7261 agatcgtcgt tttgccaggt gaccacgttg gtcaagaaat cacagccgaa gccattaagg
     7321 ttcttaaagc tatttctgat gttcgttcca atgtcaagtt cgatttcgaa aatcatttaa
     7381 ttggtggtgc tgctatcgat gctacaggtg ttccacttcc agatgaggcg ctggaagcct
     7441 ccaagaaggc tgatgccgtt ttgttaggtg ctgtgggtgg tcctaaatgg ggtaccggta
     7501 gtgttagacc tgaacaaggt ttactaaaaa tccgtaaaga acttcaattg tacgccaact
     7561 taagaccatg taactttgca tccgactctc ttttagactt atctccaatc aagccacaat
     7621 ttgctaaagg tactgacttc gttgttgtta gagaattagt gggaggtatt tactttggta
     7681 agagaaagga agacgatggt gatggtgtcg cttgggatag tgaacaatac accgttccag
     7741 aagtgcaaag aatcacaaga atggccgctt tcatggccct acaacatgag ccaccattgc
     7801 ctatttggtc cttggataaa gctaatgttt tggcctcttc aagattatgg agaaaaactg
     7861 tggaggaaac catcaagaac gaatttccta cattgaaagt tcaacatcaa ttgattgatt
     7921 ctgccgccat gatcctagtt aagaacccaa cccacctaaa tggtattata atcaccagca
     7981 acatgtttgg tgatatcatc tccgatgaag cctccgttat cccaggctcc ttgggtttgt
     8041 tgccatctgc gtccttggcc tctttgccag acaagaacac cgcatttggt ttgtacgaac
     8101 catgccacgg ttccgctcca gatttgccaa agaataaggt caaccctatc gccactatct
     8161 tgtctgctgc aatgatgttg aaattgtcat tgaacttgcc tgaagaaggt aaagccattg
     8221 aagatgcagt taaaaaggtt ttggatgcag gtatcagaac tggtgattta ggtggttcca
     8281 acagtaccac cgaagtcggt gatgctgtcg ccgaagaagt taagaaaatc cttgcttaaa
     8341 aagattctct ttttttgtga tatttgtaca taaactttat aaatgaaatt cataatagaa
     8401 acgacacgaa attacaaaat ggaatatgtt catagggtag acgaaactat atacgcaatc
     8461 tacatacatt tatcaagaag gagaaaaagg aggatgtaaa ggaatacagg taagcaaatt
     8521 gatactaatg gctcaacgtg ataaggaaaa agaattgcac tttaacatta atattgacaa
     8581 ggaggagggc atcaca
//
