LOCUS       sequence-71719-e        8328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of untagged 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 8328)
  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 8328)
  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-4XDSR
FEATURES             Location/Qualifiers
     source          1..8328
                     /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(4066..4083)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4066..4082)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4075..4097)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             6977..8071
                     /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 cttaaacaga actttaaacc
     4021 ccgggataac ttcgtatatg gtattatata cgaagttatg aattcactgg ccgtcgtttt
     4081 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     4141 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     4201 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4261 tatttcacac cgcataccat cgtccggatc aatgaagtag atgagtttac tacctgtata
     4321 tacttttttt tttgtcaaag tcacttttat ggcatttcca tgttgaagga ttagccacag
     4381 cactaaattt tcagcgtatg attgctttta aatatttaat tttcatcgtt ttttaattaa
     4441 tattcaaaac gatttaatgc cttctacatc tgaagtcaag ctcatagact cgggttgggt
     4501 agctaaaact acatctaaca cgaaggggta tagccttctt aaagtatact catgtataaa
     4561 cgttttgggt tcatattttt gggccacgtg gtttataaaa attcttaact acaccactcg
     4621 gtatcccgca cccgtctacg tttctacgat ttcgagctaa tattgagtaa agtgattagc
     4681 aaaaaaaata aaagtactcg ttgtcggaga tcaagaattt ttctattatc tcatctaaac
     4741 cactttctaa aagcgaaaaa caaaatcgta atatgcagct tgaatgggct tccatagttt
     4801 gaaagaaaaa ccctagcagt actggcaagg gagacattcc ttttacctgg ctaatcgcca
     4861 tcttccagca ggcgcaccat tgcccctgtt tcactatcca ggttacggat atagttcatg
     4921 acaatattta cattggtcca gccaccagct tgcatgatct ccggtattga aactccagcg
     4981 cgggccatat ctcgcgcggc tccgacacgg gcactgtgtc cagaccaggc caggtatctc
     5041 tgaccagagt catccttagc gccgtaaatc aatcgatgag ttgcttcaaa aatcccttcc
     5101 agggcgcgag ttgatagctg gctggtggca gatggcgcgg caacaccatt ttttctgacc
     5161 cggcaaaaca ggtagttatt cggatcatca gctacaccag agacggaaat ccatcgctcg
     5221 accagtttag ttacccccag gctaagtgcc ttctctacac ctgcggtgct aaccagcgtt
     5281 ttcgttctgc caatatggat taacattctc ccaccgtcag tacgtgagat atctttaacc
     5341 ctgatcctgg caatttcggc tatacgtaac agggtgttat aagcaatccc cagaaatgcc
     5401 agattacgta tatcctggca gcgatcgcta ttttccatga gtgaacgaac ctggtcgaaa
     5461 tcagtgcgtt cgaacgctag agcctgtttt gcacgttcac cggcatcaac gttttctttt
     5521 cggatccgcc gcataaccag tgaaacagca ttgctgtcac ttggtcgtgg cagcccggac
     5581 cgacgatgaa gcatgtttag ctggcccaaa tgttgctgga tagtttttac tgccagaccg
     5641 cgcgcctgaa gatatagaag ataatcgcga acatcttcag gttctgcggg aaaccatttc
     5701 cggttattca acttgcacca tgccgcccac gaccggcaaa cggacagaag cattttccag
     5761 gtatgctcag aaaacgcctg gcgatccctg aacatgtcca tcaggttctt gcgaacctca
     5821 tcactcgttg catcgaccgg taacttttag attaacattt gtttaaatat gaaattaggc
     5881 aatgtaacat actgcaggca aattttggtg tacggtaagt aaattggaca taagatttaa
     5941 caaagcgact ataagtcaga aagtgagaat gagattgaaa taattaattc aacttattca
     6001 attgatgata tgccaggatt cctcttcctt tatcttcaat gtttcgatta acaggtccat
     6061 tatccggttt ttagtttcgc tgcacattgc cgaatgacaa tcggattcct ttaagctatg
     6121 cttttgccct gttttctctc cccagtaatc gttgggtgac tgaaccatcc agtagccgag
     6181 agttacgttt ttcctgattt aataaatatc catatctatt tgtctcgtgg tgttttacag
     6241 caactaaccg aaattactat tttgcatcat ccaaccataa agagacaaca ataattctag
     6301 aaaacatata acaagtgttt ttttctattt acgtctcata cgcaaagtcc actttaccat
     6361 gttttggaaa cttgtaccca accattcatt tttcgatttt gtaatgaggc tcttttcttc
     6421 taacaatcat tgaggtcatt atcatgtgtc ctcacctatt gtttaaattg aggcttgcgc
     6481 tattcccaaa attgtgggta ttattttcat atatcagtat ctgcaatttc ggttcaactc
     6541 cggaagctcg gtacccaact gtgggaatac tcaggtatcg taagatgcaa gagttcgaat
     6601 ctcttagcaa ccattatttt ttttctctca acataacgag aacacacagg ggcgctatcg
     6661 cacagaatca aattcgatga ctggaaattt tttgttaatt tcagaggtcg cctgacgcat
     6721 ataccttttt caactgaaaa attgggagaa aaaggaaagg tgagagcgcc ggaaccggct
     6781 tttcatatag aatagagaag cgttcatgac taaatgcttg catcacaata cttgaagttg
     6841 acaatattat ttaaggacct attgtttttt ccaataggtg gttagcaatc gtcttacttt
     6901 ctaacttttc ttacctttta catttcagca atatatatat atatatatat atatatttca
     6961 aggatatacc attgtaatgt ctgcccctaa gaagatcgtc gttttgccag gtgaccacgt
     7021 tggtcaagaa atcacagccg aagccattaa ggttcttaaa gctatttctg atgttcgttc
     7081 caatgtcaag ttcgatttcg aaaatcattt aattggtggt gctgctatcg atgctacagg
     7141 tgttccactt ccagatgagg cgctggaagc ctccaagaag gctgatgccg ttttgttagg
     7201 tgctgtgggt ggtcctaaat ggggtaccgg tagtgttaga cctgaacaag gtttactaaa
     7261 aatccgtaaa gaacttcaat tgtacgccaa cttaagacca tgtaactttg catccgactc
     7321 tcttttagac ttatctccaa tcaagccaca atttgctaaa ggtactgact tcgttgttgt
     7381 tagagaatta gtgggaggta tttactttgg taagagaaag gaagacgatg gtgatggtgt
     7441 cgcttgggat agtgaacaat acaccgttcc agaagtgcaa agaatcacaa gaatggccgc
     7501 tttcatggcc ctacaacatg agccaccatt gcctatttgg tccttggata aagctaatgt
     7561 tttggcctct tcaagattat ggagaaaaac tgtggaggaa accatcaaga acgaatttcc
     7621 tacattgaaa gttcaacatc aattgattga ttctgccgcc atgatcctag ttaagaaccc
     7681 aacccaccta aatggtatta taatcaccag caacatgttt ggtgatatca tctccgatga
     7741 agcctccgtt atcccaggct ccttgggttt gttgccatct gcgtccttgg cctctttgcc
     7801 agacaagaac accgcatttg gtttgtacga accatgccac ggttccgctc cagatttgcc
     7861 aaagaataag gtcaacccta tcgccactat cttgtctgct gcaatgatgt tgaaattgtc
     7921 attgaacttg cctgaagaag gtaaagccat tgaagatgca gttaaaaagg ttttggatgc
     7981 aggtatcaga actggtgatt taggtggttc caacagtacc accgaagtcg gtgatgctgt
     8041 cgccgaagaa gttaagaaaa tccttgctta aaaagattct ctttttttgt gatatttgta
     8101 cataaacttt ataaatgaaa ttcataatag aaacgacacg aaattacaaa atggaatatg
     8161 ttcatagggt agacgaaact atatacgcaa tctacataca tttatcaaga aggagaaaaa
     8221 ggaggatgta aaggaataca ggtaagcaaa ttgatactaa tggctcaacg tgataaggaa
     8281 aaagaattgc actttaacat taatattgac aaggaggagg gcatcaca
//
