LOCUS       sequence-71776-e        8433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of C-terminal 3XHA tagged gene 
            sequences into the S. pombe urg1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8433)
  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 8433)
  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
FEATURES             Location/Qualifiers
     source          1..8433
                     /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(4171..4188)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4171..4187)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4180..4202)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7082..8176
                     /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 ataacttcgt
     4141 atatggtatt atatacgaag ttatgaattc actggccgtc gttttacaac gtcgtgactg
     4201 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     4261 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     4321 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4381 accatcgtcc ggatcaatga agtagatgag tttactacct gtatatactt ttttttttgt
     4441 caaagtcact tttatggcat ttccatgttg aaggattagc cacagcacta aattttcagc
     4501 gtatgattgc ttttaaatat ttaattttca tcgtttttta attaatattc aaaacgattt
     4561 aatgccttct acatctgaag tcaagctcat agactcgggt tgggtagcta aaactacatc
     4621 taacacgaag gggtatagcc ttcttaaagt atactcatgt ataaacgttt tgggttcata
     4681 tttttgggcc acgtggttta taaaaattct taactacacc actcggtatc ccgcacccgt
     4741 ctacgtttct acgatttcga gctaatattg agtaaagtga ttagcaaaaa aaataaaagt
     4801 actcgttgtc ggagatcaag aatttttcta ttatctcatc taaaccactt tctaaaagcg
     4861 aaaaacaaaa tcgtaatatg cagcttgaat gggcttccat agtttgaaag aaaaacccta
     4921 gcagtactgg caagggagac attcctttta cctggctaat cgccatcttc cagcaggcgc
     4981 accattgccc ctgtttcact atccaggtta cggatatagt tcatgacaat atttacattg
     5041 gtccagccac cagcttgcat gatctccggt attgaaactc cagcgcgggc catatctcgc
     5101 gcggctccga cacgggcact gtgtccagac caggccaggt atctctgacc agagtcatcc
     5161 ttagcgccgt aaatcaatcg atgagttgct tcaaaaatcc cttccagggc gcgagttgat
     5221 agctggctgg tggcagatgg cgcggcaaca ccattttttc tgacccggca aaacaggtag
     5281 ttattcggat catcagctac accagagacg gaaatccatc gctcgaccag tttagttacc
     5341 cccaggctaa gtgccttctc tacacctgcg gtgctaacca gcgttttcgt tctgccaata
     5401 tggattaaca ttctcccacc gtcagtacgt gagatatctt taaccctgat cctggcaatt
     5461 tcggctatac gtaacagggt gttataagca atccccagaa atgccagatt acgtatatcc
     5521 tggcagcgat cgctattttc catgagtgaa cgaacctggt cgaaatcagt gcgttcgaac
     5581 gctagagcct gttttgcacg ttcaccggca tcaacgtttt cttttcggat ccgccgcata
     5641 accagtgaaa cagcattgct gtcacttggt cgtggcagcc cggaccgacg atgaagcatg
     5701 tttagctggc ccaaatgttg ctggatagtt tttactgcca gaccgcgcgc ctgaagatat
     5761 agaagataat cgcgaacatc ttcaggttct gcgggaaacc atttccggtt attcaacttg
     5821 caccatgccg cccacgaccg gcaaacggac agaagcattt tccaggtatg ctcagaaaac
     5881 gcctggcgat ccctgaacat gtccatcagg ttcttgcgaa cctcatcact cgttgcatcg
     5941 accggtaact tttagattaa catttgttta aatatgaaat taggcaatgt aacatactgc
     6001 aggcaaattt tggtgtacgg taagtaaatt ggacataaga tttaacaaag cgactataag
     6061 tcagaaagtg agaatgagat tgaaataatt aattcaactt attcaattga tgatatgcca
     6121 ggattcctct tcctttatct tcaatgtttc gattaacagg tccattatcc ggtttttagt
     6181 ttcgctgcac attgccgaat gacaatcgga ttcctttaag ctatgctttt gccctgtttt
     6241 ctctccccag taatcgttgg gtgactgaac catccagtag ccgagagtta cgtttttcct
     6301 gatttaataa atatccatat ctatttgtct cgtggtgttt tacagcaact aaccgaaatt
     6361 actattttgc atcatccaac cataaagaga caacaataat tctagaaaac atataacaag
     6421 tgtttttttc tatttacgtc tcatacgcaa agtccacttt accatgtttt ggaaacttgt
     6481 acccaaccat tcatttttcg attttgtaat gaggctcttt tcttctaaca atcattgagg
     6541 tcattatcat gtgtcctcac ctattgttta aattgaggct tgcgctattc ccaaaattgt
     6601 gggtattatt ttcatatatc agtatctgca atttcggttc aactccggaa gctcggtacc
     6661 caactgtggg aatactcagg tatcgtaaga tgcaagagtt cgaatctctt agcaaccatt
     6721 attttttttc tctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc
     6781 gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact
     6841 gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag
     6901 agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat attatttaag
     6961 gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac ttttcttacc
     7021 ttttacattt cagcaatata tatatatata tatatatata tttcaaggat ataccattgt
     7081 aatgtctgcc cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc aagaaatcac
     7141 agccgaagcc attaaggttc ttaaagctat ttctgatgtt cgttccaatg tcaagttcga
     7201 tttcgaaaat catttaattg gtggtgctgc tatcgatgct acaggtgttc cacttccaga
     7261 tgaggcgctg gaagcctcca agaaggctga tgccgttttg ttaggtgctg tgggtggtcc
     7321 taaatggggt accggtagtg ttagacctga acaaggttta ctaaaaatcc gtaaagaact
     7381 tcaattgtac gccaacttaa gaccatgtaa ctttgcatcc gactctcttt tagacttatc
     7441 tccaatcaag ccacaatttg ctaaaggtac tgacttcgtt gttgttagag aattagtggg
     7501 aggtatttac tttggtaaga gaaaggaaga cgatggtgat ggtgtcgctt gggatagtga
     7561 acaatacacc gttccagaag tgcaaagaat cacaagaatg gccgctttca tggccctaca
     7621 acatgagcca ccattgccta tttggtcctt ggataaagct aatgttttgg cctcttcaag
     7681 attatggaga aaaactgtgg aggaaaccat caagaacgaa tttcctacat tgaaagttca
     7741 acatcaattg attgattctg ccgccatgat cctagttaag aacccaaccc acctaaatgg
     7801 tattataatc accagcaaca tgtttggtga tatcatctcc gatgaagcct ccgttatccc
     7861 aggctccttg ggtttgttgc catctgcgtc cttggcctct ttgccagaca agaacaccgc
     7921 atttggtttg tacgaaccat gccacggttc cgctccagat ttgccaaaga ataaggtcaa
     7981 ccctatcgcc actatcttgt ctgctgcaat gatgttgaaa ttgtcattga acttgcctga
     8041 agaaggtaaa gccattgaag atgcagttaa aaaggttttg gatgcaggta tcagaactgg
     8101 tgatttaggt ggttccaaca gtaccaccga agtcggtgat gctgtcgccg aagaagttaa
     8161 gaaaatcctt gcttaaaaag attctctttt tttgtgatat ttgtacataa actttataaa
     8221 tgaaattcat aatagaaacg acacgaaatt acaaaatgga atatgttcat agggtagacg
     8281 aaactatata cgcaatctac atacatttat caagaaggag aaaaaggagg atgtaaagga
     8341 atacaggtaa gcaaattgat actaatggct caacgtgata aggaaaaaga attgcacttt
     8401 aacattaata ttgacaagga ggagggcatc aca
//
