LOCUS       sequence-74368-e        8499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of C-terminal 3XHA tagged 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 8499)
  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 8499)
  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-6XDSR
FEATURES             Location/Qualifiers
     source          1..8499
                     /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(4237..4254)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4237..4253)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4246..4268)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             7148..8242
                     /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 atctttaaac tccgtattaa
     4141 acccattctt aaacagaact ttaaacggca ggttaaacgt aatgttaaac cccgggataa
     4201 cttcgtatat ggtattatat acgaagttat gaattcactg gccgtcgttt tacaacgtcg
     4261 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4321 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4381 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4441 ccgcatacca tcgtccggat caatgaagta gatgagttta ctacctgtat atactttttt
     4501 ttttgtcaaa gtcactttta tggcatttcc atgttgaagg attagccaca gcactaaatt
     4561 ttcagcgtat gattgctttt aaatatttaa ttttcatcgt tttttaatta atattcaaaa
     4621 cgatttaatg ccttctacat ctgaagtcaa gctcatagac tcgggttggg tagctaaaac
     4681 tacatctaac acgaaggggt atagccttct taaagtatac tcatgtataa acgttttggg
     4741 ttcatatttt tgggccacgt ggtttataaa aattcttaac tacaccactc ggtatcccgc
     4801 acccgtctac gtttctacga tttcgagcta atattgagta aagtgattag caaaaaaaat
     4861 aaaagtactc gttgtcggag atcaagaatt tttctattat ctcatctaaa ccactttcta
     4921 aaagcgaaaa acaaaatcgt aatatgcagc ttgaatgggc ttccatagtt tgaaagaaaa
     4981 accctagcag tactggcaag ggagacattc cttttacctg gctaatcgcc atcttccagc
     5041 aggcgcacca ttgcccctgt ttcactatcc aggttacgga tatagttcat gacaatattt
     5101 acattggtcc agccaccagc ttgcatgatc tccggtattg aaactccagc gcgggccata
     5161 tctcgcgcgg ctccgacacg ggcactgtgt ccagaccagg ccaggtatct ctgaccagag
     5221 tcatccttag cgccgtaaat caatcgatga gttgcttcaa aaatcccttc cagggcgcga
     5281 gttgatagct ggctggtggc agatggcgcg gcaacaccat tttttctgac ccggcaaaac
     5341 aggtagttat tcggatcatc agctacacca gagacggaaa tccatcgctc gaccagttta
     5401 gttaccccca ggctaagtgc cttctctaca cctgcggtgc taaccagcgt tttcgttctg
     5461 ccaatatgga ttaacattct cccaccgtca gtacgtgaga tatctttaac cctgatcctg
     5521 gcaatttcgg ctatacgtaa cagggtgtta taagcaatcc ccagaaatgc cagattacgt
     5581 atatcctggc agcgatcgct attttccatg agtgaacgaa cctggtcgaa atcagtgcgt
     5641 tcgaacgcta gagcctgttt tgcacgttca ccggcatcaa cgttttcttt tcggatccgc
     5701 cgcataacca gtgaaacagc attgctgtca cttggtcgtg gcagcccgga ccgacgatga
     5761 agcatgttta gctggcccaa atgttgctgg atagttttta ctgccagacc gcgcgcctga
     5821 agatatagaa gataatcgcg aacatcttca ggttctgcgg gaaaccattt ccggttattc
     5881 aacttgcacc atgccgccca cgaccggcaa acggacagaa gcattttcca ggtatgctca
     5941 gaaaacgcct ggcgatccct gaacatgtcc atcaggttct tgcgaacctc atcactcgtt
     6001 gcatcgaccg gtaactttta gattaacatt tgtttaaata tgaaattagg caatgtaaca
     6061 tactgcaggc aaattttggt gtacggtaag taaattggac ataagattta acaaagcgac
     6121 tataagtcag aaagtgagaa tgagattgaa ataattaatt caacttattc aattgatgat
     6181 atgccaggat tcctcttcct ttatcttcaa tgtttcgatt aacaggtcca ttatccggtt
     6241 tttagtttcg ctgcacattg ccgaatgaca atcggattcc tttaagctat gcttttgccc
     6301 tgttttctct ccccagtaat cgttgggtga ctgaaccatc cagtagccga gagttacgtt
     6361 tttcctgatt taataaatat ccatatctat ttgtctcgtg gtgttttaca gcaactaacc
     6421 gaaattacta ttttgcatca tccaaccata aagagacaac aataattcta gaaaacatat
     6481 aacaagtgtt tttttctatt tacgtctcat acgcaaagtc cactttacca tgttttggaa
     6541 acttgtaccc aaccattcat ttttcgattt tgtaatgagg ctcttttctt ctaacaatca
     6601 ttgaggtcat tatcatgtgt cctcacctat tgtttaaatt gaggcttgcg ctattcccaa
     6661 aattgtgggt attattttca tatatcagta tctgcaattt cggttcaact ccggaagctc
     6721 ggtacccaac tgtgggaata ctcaggtatc gtaagatgca agagttcgaa tctcttagca
     6781 accattattt tttttctctc aacataacga gaacacacag gggcgctatc gcacagaatc
     6841 aaattcgatg actggaaatt ttttgttaat ttcagaggtc gcctgacgca tatacctttt
     6901 tcaactgaaa aattgggaga aaaaggaaag gtgagagcgc cggaaccggc ttttcatata
     6961 gaatagagaa gcgttcatga ctaaatgctt gcatcacaat acttgaagtt gacaatatta
     7021 tttaaggacc tattgttttt tccaataggt ggttagcaat cgtcttactt tctaactttt
     7081 cttacctttt acatttcagc aatatatata tatatatata tatatatttc aaggatatac
     7141 cattgtaatg tctgccccta agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga
     7201 aatcacagcc gaagccatta aggttcttaa agctatttct gatgttcgtt ccaatgtcaa
     7261 gttcgatttc gaaaatcatt taattggtgg tgctgctatc gatgctacag gtgttccact
     7321 tccagatgag gcgctggaag cctccaagaa ggctgatgcc gttttgttag gtgctgtggg
     7381 tggtcctaaa tggggtaccg gtagtgttag acctgaacaa ggtttactaa aaatccgtaa
     7441 agaacttcaa ttgtacgcca acttaagacc atgtaacttt gcatccgact ctcttttaga
     7501 cttatctcca atcaagccac aatttgctaa aggtactgac ttcgttgttg ttagagaatt
     7561 agtgggaggt atttactttg gtaagagaaa ggaagacgat ggtgatggtg tcgcttggga
     7621 tagtgaacaa tacaccgttc cagaagtgca aagaatcaca agaatggccg ctttcatggc
     7681 cctacaacat gagccaccat tgcctatttg gtccttggat aaagctaatg ttttggcctc
     7741 ttcaagatta tggagaaaaa ctgtggagga aaccatcaag aacgaatttc ctacattgaa
     7801 agttcaacat caattgattg attctgccgc catgatccta gttaagaacc caacccacct
     7861 aaatggtatt ataatcacca gcaacatgtt tggtgatatc atctccgatg aagcctccgt
     7921 tatcccaggc tccttgggtt tgttgccatc tgcgtccttg gcctctttgc cagacaagaa
     7981 caccgcattt ggtttgtacg aaccatgcca cggttccgct ccagatttgc caaagaataa
     8041 ggtcaaccct atcgccacta tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt
     8101 gcctgaagaa ggtaaagcca ttgaagatgc agttaaaaag gttttggatg caggtatcag
     8161 aactggtgat ttaggtggtt ccaacagtac caccgaagtc ggtgatgctg tcgccgaaga
     8221 agttaagaaa atccttgctt aaaaagattc tctttttttg tgatatttgt acataaactt
     8281 tataaatgaa attcataata gaaacgacac gaaattacaa aatggaatat gttcataggg
     8341 tagacgaaac tatatacgca atctacatac atttatcaag aaggagaaaa aggaggatgt
     8401 aaaggaatac aggtaagcaa attgatacta atggctcaac gtgataagga aaaagaattg
     8461 cactttaaca ttaatattga caaggaggag ggcatcaca
//
