LOCUS       sequence-71714-e        8316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of untagged gene sequences together 
            with 3 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 8316)
  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 8316)
  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-3XDSR
FEATURES             Location/Qualifiers
     source          1..8316
                     /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)."
     primer_bind     complement(4054..4071)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4054..4070)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4063..4085)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             6965..8059
                     /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 gactagttta aactccgtat taaacccatt cttaaacccc gggataactt
     4021 cgtatatggt attatatacg aagttatgaa ttcactggcc gtcgttttac aacgtcgtga
     4081 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4141 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4201 tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4261 cataccatcg tccggatcaa tgaagtagat gagtttacta cctgtatata cttttttttt
     4321 tgtcaaagtc acttttatgg catttccatg ttgaaggatt agccacagca ctaaattttc
     4381 agcgtatgat tgcttttaaa tatttaattt tcatcgtttt ttaattaata ttcaaaacga
     4441 tttaatgcct tctacatctg aagtcaagct catagactcg ggttgggtag ctaaaactac
     4501 atctaacacg aaggggtata gccttcttaa agtatactca tgtataaacg ttttgggttc
     4561 atatttttgg gccacgtggt ttataaaaat tcttaactac accactcggt atcccgcacc
     4621 cgtctacgtt tctacgattt cgagctaata ttgagtaaag tgattagcaa aaaaaataaa
     4681 agtactcgtt gtcggagatc aagaattttt ctattatctc atctaaacca ctttctaaaa
     4741 gcgaaaaaca aaatcgtaat atgcagcttg aatgggcttc catagtttga aagaaaaacc
     4801 ctagcagtac tggcaaggga gacattcctt ttacctggct aatcgccatc ttccagcagg
     4861 cgcaccattg cccctgtttc actatccagg ttacggatat agttcatgac aatatttaca
     4921 ttggtccagc caccagcttg catgatctcc ggtattgaaa ctccagcgcg ggccatatct
     4981 cgcgcggctc cgacacgggc actgtgtcca gaccaggcca ggtatctctg accagagtca
     5041 tccttagcgc cgtaaatcaa tcgatgagtt gcttcaaaaa tcccttccag ggcgcgagtt
     5101 gatagctggc tggtggcaga tggcgcggca acaccatttt ttctgacccg gcaaaacagg
     5161 tagttattcg gatcatcagc tacaccagag acggaaatcc atcgctcgac cagtttagtt
     5221 acccccaggc taagtgcctt ctctacacct gcggtgctaa ccagcgtttt cgttctgcca
     5281 atatggatta acattctccc accgtcagta cgtgagatat ctttaaccct gatcctggca
     5341 atttcggcta tacgtaacag ggtgttataa gcaatcccca gaaatgccag attacgtata
     5401 tcctggcagc gatcgctatt ttccatgagt gaacgaacct ggtcgaaatc agtgcgttcg
     5461 aacgctagag cctgttttgc acgttcaccg gcatcaacgt tttcttttcg gatccgccgc
     5521 ataaccagtg aaacagcatt gctgtcactt ggtcgtggca gcccggaccg acgatgaagc
     5581 atgtttagct ggcccaaatg ttgctggata gtttttactg ccagaccgcg cgcctgaaga
     5641 tatagaagat aatcgcgaac atcttcaggt tctgcgggaa accatttccg gttattcaac
     5701 ttgcaccatg ccgcccacga ccggcaaacg gacagaagca ttttccaggt atgctcagaa
     5761 aacgcctggc gatccctgaa catgtccatc aggttcttgc gaacctcatc actcgttgca
     5821 tcgaccggta acttttagat taacatttgt ttaaatatga aattaggcaa tgtaacatac
     5881 tgcaggcaaa ttttggtgta cggtaagtaa attggacata agatttaaca aagcgactat
     5941 aagtcagaaa gtgagaatga gattgaaata attaattcaa cttattcaat tgatgatatg
     6001 ccaggattcc tcttccttta tcttcaatgt ttcgattaac aggtccatta tccggttttt
     6061 agtttcgctg cacattgccg aatgacaatc ggattccttt aagctatgct tttgccctgt
     6121 tttctctccc cagtaatcgt tgggtgactg aaccatccag tagccgagag ttacgttttt
     6181 cctgatttaa taaatatcca tatctatttg tctcgtggtg ttttacagca actaaccgaa
     6241 attactattt tgcatcatcc aaccataaag agacaacaat aattctagaa aacatataac
     6301 aagtgttttt ttctatttac gtctcatacg caaagtccac tttaccatgt tttggaaact
     6361 tgtacccaac cattcatttt tcgattttgt aatgaggctc ttttcttcta acaatcattg
     6421 aggtcattat catgtgtcct cacctattgt ttaaattgag gcttgcgcta ttcccaaaat
     6481 tgtgggtatt attttcatat atcagtatct gcaatttcgg ttcaactccg gaagctcggt
     6541 acccaactgt gggaatactc aggtatcgta agatgcaaga gttcgaatct cttagcaacc
     6601 attatttttt ttctctcaac ataacgagaa cacacagggg cgctatcgca cagaatcaaa
     6661 ttcgatgact ggaaattttt tgttaatttc agaggtcgcc tgacgcatat acctttttca
     6721 actgaaaaat tgggagaaaa aggaaaggtg agagcgccgg aaccggcttt tcatatagaa
     6781 tagagaagcg ttcatgacta aatgcttgca tcacaatact tgaagttgac aatattattt
     6841 aaggacctat tgttttttcc aataggtggt tagcaatcgt cttactttct aacttttctt
     6901 accttttaca tttcagcaat atatatatat atatatatat atatttcaag gatataccat
     6961 tgtaatgtct gcccctaaga agatcgtcgt tttgccaggt gaccacgttg gtcaagaaat
     7021 cacagccgaa gccattaagg ttcttaaagc tatttctgat gttcgttcca atgtcaagtt
     7081 cgatttcgaa aatcatttaa ttggtggtgc tgctatcgat gctacaggtg ttccacttcc
     7141 agatgaggcg ctggaagcct ccaagaaggc tgatgccgtt ttgttaggtg ctgtgggtgg
     7201 tcctaaatgg ggtaccggta gtgttagacc tgaacaaggt ttactaaaaa tccgtaaaga
     7261 acttcaattg tacgccaact taagaccatg taactttgca tccgactctc ttttagactt
     7321 atctccaatc aagccacaat ttgctaaagg tactgacttc gttgttgtta gagaattagt
     7381 gggaggtatt tactttggta agagaaagga agacgatggt gatggtgtcg cttgggatag
     7441 tgaacaatac accgttccag aagtgcaaag aatcacaaga atggccgctt tcatggccct
     7501 acaacatgag ccaccattgc ctatttggtc cttggataaa gctaatgttt tggcctcttc
     7561 aagattatgg agaaaaactg tggaggaaac catcaagaac gaatttccta cattgaaagt
     7621 tcaacatcaa ttgattgatt ctgccgccat gatcctagtt aagaacccaa cccacctaaa
     7681 tggtattata atcaccagca acatgtttgg tgatatcatc tccgatgaag cctccgttat
     7741 cccaggctcc ttgggtttgt tgccatctgc gtccttggcc tctttgccag acaagaacac
     7801 cgcatttggt ttgtacgaac catgccacgg ttccgctcca gatttgccaa agaataaggt
     7861 caaccctatc gccactatct tgtctgctgc aatgatgttg aaattgtcat tgaacttgcc
     7921 tgaagaaggt aaagccattg aagatgcagt taaaaaggtt ttggatgcag gtatcagaac
     7981 tggtgattta ggtggttcca acagtaccac cgaagtcggt gatgctgtcg ccgaagaagt
     8041 taagaaaatc cttgcttaaa aagattctct ttttttgtga tatttgtaca taaactttat
     8101 aaatgaaatt cataatagaa acgacacgaa attacaaaat ggaatatgtt catagggtag
     8161 acgaaactat atacgcaatc tacatacatt tatcaagaag gagaaaaagg aggatgtaaa
     8221 ggaatacagg taagcaaatt gatactaatg gctcaacgtg ataaggaaaa agaattgcac
     8281 tttaacatta atattgacaa ggaggagggc atcaca
//
