LOCUS       sequence-71721-e        8352 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of untagged gene sequences together 
            with 6 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 8352)
  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 8352)
  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-6XDSR
FEATURES             Location/Qualifiers
     source          1..8352
                     /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(4090..4107)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4090..4106)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4099..4121)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7001..8095
                     /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 actttaaacg
     4021 gcaggttaaa cgtaatgtta aaccccggga taacttcgta tatggtatta tatacgaagt
     4081 tatgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     4141 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     4201 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     4261 ttttctcctt acgcatctgt gcggtatttc acaccgcata ccatcgtccg gatcaatgaa
     4321 gtagatgagt ttactacctg tatatacttt tttttttgtc aaagtcactt ttatggcatt
     4381 tccatgttga aggattagcc acagcactaa attttcagcg tatgattgct tttaaatatt
     4441 taattttcat cgttttttaa ttaatattca aaacgattta atgccttcta catctgaagt
     4501 caagctcata gactcgggtt gggtagctaa aactacatct aacacgaagg ggtatagcct
     4561 tcttaaagta tactcatgta taaacgtttt gggttcatat ttttgggcca cgtggtttat
     4621 aaaaattctt aactacacca ctcggtatcc cgcacccgtc tacgtttcta cgatttcgag
     4681 ctaatattga gtaaagtgat tagcaaaaaa aataaaagta ctcgttgtcg gagatcaaga
     4741 atttttctat tatctcatct aaaccacttt ctaaaagcga aaaacaaaat cgtaatatgc
     4801 agcttgaatg ggcttccata gtttgaaaga aaaaccctag cagtactggc aagggagaca
     4861 ttccttttac ctggctaatc gccatcttcc agcaggcgca ccattgcccc tgtttcacta
     4921 tccaggttac ggatatagtt catgacaata tttacattgg tccagccacc agcttgcatg
     4981 atctccggta ttgaaactcc agcgcgggcc atatctcgcg cggctccgac acgggcactg
     5041 tgtccagacc aggccaggta tctctgacca gagtcatcct tagcgccgta aatcaatcga
     5101 tgagttgctt caaaaatccc ttccagggcg cgagttgata gctggctggt ggcagatggc
     5161 gcggcaacac cattttttct gacccggcaa aacaggtagt tattcggatc atcagctaca
     5221 ccagagacgg aaatccatcg ctcgaccagt ttagttaccc ccaggctaag tgccttctct
     5281 acacctgcgg tgctaaccag cgttttcgtt ctgccaatat ggattaacat tctcccaccg
     5341 tcagtacgtg agatatcttt aaccctgatc ctggcaattt cggctatacg taacagggtg
     5401 ttataagcaa tccccagaaa tgccagatta cgtatatcct ggcagcgatc gctattttcc
     5461 atgagtgaac gaacctggtc gaaatcagtg cgttcgaacg ctagagcctg ttttgcacgt
     5521 tcaccggcat caacgttttc ttttcggatc cgccgcataa ccagtgaaac agcattgctg
     5581 tcacttggtc gtggcagccc ggaccgacga tgaagcatgt ttagctggcc caaatgttgc
     5641 tggatagttt ttactgccag accgcgcgcc tgaagatata gaagataatc gcgaacatct
     5701 tcaggttctg cgggaaacca tttccggtta ttcaacttgc accatgccgc ccacgaccgg
     5761 caaacggaca gaagcatttt ccaggtatgc tcagaaaacg cctggcgatc cctgaacatg
     5821 tccatcaggt tcttgcgaac ctcatcactc gttgcatcga ccggtaactt ttagattaac
     5881 atttgtttaa atatgaaatt aggcaatgta acatactgca ggcaaatttt ggtgtacggt
     5941 aagtaaattg gacataagat ttaacaaagc gactataagt cagaaagtga gaatgagatt
     6001 gaaataatta attcaactta ttcaattgat gatatgccag gattcctctt cctttatctt
     6061 caatgtttcg attaacaggt ccattatccg gtttttagtt tcgctgcaca ttgccgaatg
     6121 acaatcggat tcctttaagc tatgcttttg ccctgttttc tctccccagt aatcgttggg
     6181 tgactgaacc atccagtagc cgagagttac gtttttcctg atttaataaa tatccatatc
     6241 tatttgtctc gtggtgtttt acagcaacta accgaaatta ctattttgca tcatccaacc
     6301 ataaagagac aacaataatt ctagaaaaca tataacaagt gtttttttct atttacgtct
     6361 catacgcaaa gtccacttta ccatgttttg gaaacttgta cccaaccatt catttttcga
     6421 ttttgtaatg aggctctttt cttctaacaa tcattgaggt cattatcatg tgtcctcacc
     6481 tattgtttaa attgaggctt gcgctattcc caaaattgtg ggtattattt tcatatatca
     6541 gtatctgcaa tttcggttca actccggaag ctcggtaccc aactgtggga atactcaggt
     6601 atcgtaagat gcaagagttc gaatctctta gcaaccatta ttttttttct ctcaacataa
     6661 cgagaacaca caggggcgct atcgcacaga atcaaattcg atgactggaa attttttgtt
     6721 aatttcagag gtcgcctgac gcatatacct ttttcaactg aaaaattggg agaaaaagga
     6781 aaggtgagag cgccggaacc ggcttttcat atagaataga gaagcgttca tgactaaatg
     6841 cttgcatcac aatacttgaa gttgacaata ttatttaagg acctattgtt ttttccaata
     6901 ggtggttagc aatcgtctta ctttctaact tttcttacct tttacatttc agcaatatat
     6961 atatatatat atatatatat ttcaaggata taccattgta atgtctgccc ctaagaagat
     7021 cgtcgttttg ccaggtgacc acgttggtca agaaatcaca gccgaagcca ttaaggttct
     7081 taaagctatt tctgatgttc gttccaatgt caagttcgat ttcgaaaatc atttaattgg
     7141 tggtgctgct atcgatgcta caggtgttcc acttccagat gaggcgctgg aagcctccaa
     7201 gaaggctgat gccgttttgt taggtgctgt gggtggtcct aaatggggta ccggtagtgt
     7261 tagacctgaa caaggtttac taaaaatccg taaagaactt caattgtacg ccaacttaag
     7321 accatgtaac tttgcatccg actctctttt agacttatct ccaatcaagc cacaatttgc
     7381 taaaggtact gacttcgttg ttgttagaga attagtggga ggtatttact ttggtaagag
     7441 aaaggaagac gatggtgatg gtgtcgcttg ggatagtgaa caatacaccg ttccagaagt
     7501 gcaaagaatc acaagaatgg ccgctttcat ggccctacaa catgagccac cattgcctat
     7561 ttggtccttg gataaagcta atgttttggc ctcttcaaga ttatggagaa aaactgtgga
     7621 ggaaaccatc aagaacgaat ttcctacatt gaaagttcaa catcaattga ttgattctgc
     7681 cgccatgatc ctagttaaga acccaaccca cctaaatggt attataatca ccagcaacat
     7741 gtttggtgat atcatctccg atgaagcctc cgttatccca ggctccttgg gtttgttgcc
     7801 atctgcgtcc ttggcctctt tgccagacaa gaacaccgca tttggtttgt acgaaccatg
     7861 ccacggttcc gctccagatt tgccaaagaa taaggtcaac cctatcgcca ctatcttgtc
     7921 tgctgcaatg atgttgaaat tgtcattgaa cttgcctgaa gaaggtaaag ccattgaaga
     7981 tgcagttaaa aaggttttgg atgcaggtat cagaactggt gatttaggtg gttccaacag
     8041 taccaccgaa gtcggtgatg ctgtcgccga agaagttaag aaaatccttg cttaaaaaga
     8101 ttctcttttt ttgtgatatt tgtacataaa ctttataaat gaaattcata atagaaacga
     8161 cacgaaatta caaaatggaa tatgttcata gggtagacga aactatatac gcaatctaca
     8221 tacatttatc aagaaggaga aaaaggagga tgtaaaggaa tacaggtaag caaattgata
     8281 ctaatggctc aacgtgataa ggaaaaagaa ttgcacttta acattaatat tgacaaggag
     8341 gagggcatca ca
//
