LOCUS       sequence-182259-        8523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  genomic GBP-mCherry epitope C-terminal tagging at leu1+ locus in S. 
            pombe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8523)
  AUTHORS   Chen YH, Wang GY, Hao HC, Chao CJ, Wang Y, Jin QW
  TITLE     Facile manipulation of protein localization in fission yeast through
            GBP-GFP binding.
  JOURNAL   J Cell Sci. 2017 Jan 12. pii: jcs.198457. doi: 10.1242/jcs.198457.
  PUBMED    28082423
REFERENCE   2  (bases 1 to 8523)
  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: "J Cell Sci.
            2017 Jan 12. pii: jcs.198457. doi: 10.1242/jcs.198457."
COMMENT     Sequence Label: pJK148-Pnmt41-GBP-mCherry(C)-leu1+
FEATURES             Location/Qualifiers
     source          1..8523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2303..2758)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2440..2461)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2652..2671
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2884..2906
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2898..2915
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2899..2915
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2925..2944
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2925..2943
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2989..4143
                     /label=nmt1 P41 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring medium strength thiamine-repressible expression"
     CDS             4187..4528
                     /codon_start=1
                     /product="GFP-binding fragment of a single-chain camelid 
                     antibody (Rothbauer et al., 2008)"
                     /label=GFP nanobody
                     /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
                     WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
                     GQGTQVTVSS"
     CDS             4532..5242
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGAMDELYK"
     primer_bind     complement(4677..4695)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4945..4964
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(6263..6283)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6263..6281)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6302..6318)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6302..6318)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6315..6337)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6326..6342
                     /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)."
     promoter        complement(join(6350..6356,6357..6374,6375..6380))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6395..6416
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6533..6550)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6704..7292)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6784..6803)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7463..8254,8255..8323))
                     /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     8086..8105
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8324..8428)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8496..8514
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatacgat atcccaatct gtagtataaa aagtgcaaca gtgttgagtt tcccgaaacc
      241 aggcattttt gcggtagggt ctgaaacttt cacgaaacca tttcacaaca aaacttcttc
      301 aacaacatgt gtgcaaagaa aatcgtcgtc ttaccaggag accatattgg ccctgaaatt
      361 gttgcttctg ccttggaggt tttgaaagtc gttgagaaga agcgacctga gttaaaactc
      421 gagtttgaag aacacaagat tggaggtgcc tctattgatg cctatggaac ccctttgact
      481 gacgagactg tgaaggcttg tttggaagct gacggtgttc ttttgggtgc cgttggtggt
      541 cctgaatgga ccaaccccaa ttgtcgtcct gagcaaggtt tattgaagct tcgtaagagt
      601 atgggtgttt gggccaacct tcgaccttgc aactttgcca gcaagtcttt agtcaagtac
      661 agccctttga agcctgaaat cgttgaaggt gtcgattttt gtgttgtacg agaacttact
      721 ggaggttgtt actttggtga gcgcactgag gacaacggat cgggttatgc tatggacact
      781 tggccttaca gtttggaaga agtttctcgt attgctcgtt tggctgcttg gttagctgaa
      841 acttccaacc ctcctgctcc cgtcacatta ctcgacaaag ctaatgtttt ggcaacttct
      901 cgtctttggc gcaagactgt agctaagatt tttaaggagg aatatcctca ccttacctta
      961 aaaaccagct cattgactcc gcagccatgc ttttggtcaa gagccctcgt acacttaacg
     1021 gtgttgtttt gactgacaac ttgtttggtg acattatttc agatgaggct tctgtcattc
     1081 ctggtagctt gggccttttg ccttctgcct ccctttccgg tgtggtagga aaatcagaag
     1141 aaaaggttca ttgtttggtt gagcccattc acggtagcgc tcccgatatc gctggcaagg
     1201 gcattgttaa tcctgttggt acaattttat ctgcttccct tctccttcgt tatggtttga
     1261 atgctcctaa ggaggctgaa gctatcgaag ccgccgtacg caaggtcttg gatgatactt
     1321 caattggtgg acgtggtctt tatactcgcg atttgggagg tgaggcttct accgctgata
     1381 ttactaaggc tgttgttgaa gaacttgaaa aaattttgta atagggttta atgtagaata
     1441 aattcacatg atttgatttg acctaatttt tattgattaa ttgtgtcgca tatttattat
     1501 cgttaaaaat gcaattgaca aaagcgtaaa ttttaaggct ctaatgtttt ttcttttggt
     1561 atgttatagt caagcaaaga tgtattaagt tgcaaaagca tggaattgat ttacgacatc
     1621 cgttcaactt tatgaccgag ttgtcgctgt gtacttactc tttttttact atgaaaagtt
     1681 tcaaacataa tatcattaat tatttctttt taaacgaagt aacttgtaac ataaataata
     1741 ggggaaaaga agaaaaaaac cataaaaatt cgagaacgta acaattatta ttgtaacgat
     1801 gacaacccga ttctttgaga gagccagaga catgactttt gcattaccgt aattggtttc
     1861 tacttatgaa ttgtaaactt gtgtagggaa tttcatccac agctatcgcg ggctgctctc
     1921 tgcatcagtg ttagcacatt ttatattaat gaagattttg ctatgcaaaa tacgtacatt
     1981 tctggcgcat atatttttat caaacgaaca tgactatcct ttccactttg atggttcgca
     2041 agtattttga tttgtattaa atgcacatct aacaagccat ttcgcccaaa atcaccaact
     2101 ggcacatgta cccaaccagt tagttcctcg acttgaactg ttgtaacaat ttccaaatct
     2161 tgaaaccctg tcccagctga tattcgcaaa gtactgggag tataagattc atcaagggtg
     2221 tattgaaggt acatactcac atacttaatc gatgcggtgt gaaataccgc acagatgcgt
     2281 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     2341 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2401 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2461 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2521 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     2581 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2641 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2701 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     2761 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     2821 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     2881 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat
     2941 agggcgaatt ggctgcaggt cgatcgactc tagaggatca gaaaattatc gccataaaag
     3001 acagaataag tcatcagcgg ttgtttcatt tcctatattt tttttttatt tttttatttt
     3061 ttaataaggg aaaatttaac gtctaaggat acagaagatt gttagcacat taaagtaata
     3121 aaggcttaag tagtaagtgc cttagcatgt tattgtattt caaaggacat aatctaaaat
     3181 aataacaata tcatttctca caagttattc aattttcttt tttttttcta ataatatcaa
     3241 gaatgtatta tttgtttgac ataagtcaac taatttattt aatatgctgg attaatcttg
     3301 cagacatgta aattaacaag ttttagtcaa ataacgttga agtttcaatg aactcaaata
     3361 atttctcttt ttttttatat aaccataatc tgatttatat tttccgcagg gatcaactga
     3421 agttatgaca tttggattgg atcacttata accttggtcg ccaaataata caaaaatcag
     3481 cgttataaaa caaagaaggt ttttgttaag aaattaatcc tctttcttga taagaaagtt
     3541 gaaccgaaat tgcagatact gatatatgaa aataataccc acaattttgg gaatagcgca
     3601 agcctcaatt taaacaatag gtgaggacac atgataatga cctcaatgat tgttagaaga
     3661 aaagagcctc attacaaaat cgaaaaatga atggttgggt acaagtttcc aaaacatggt
     3721 aaagtggact ttgcgtatga gacgtaaata gaaaaaaaca cttgttatat gttttctaga
     3781 attattgttg tctctttatg gttggatgat gcaaaatagt aatttcggtt agttgctgta
     3841 aaacaccacg agacaaatag atatggatat ttattaaatc aggaaaaacg taactctcgg
     3901 ctactggatg gttcagtcac ccaacgatta ctggggagag aaaacagggc aaaagcaaag
     3961 cttaaaggaa tccgattgtc attcggcaat gtgcagcgaa actaaaaacc ggataatgga
     4021 cctgttaatc gaaacattga agataaagga agaggaatcc tggcatatca tcaattgaat
     4081 aagttgaatt aattatttca atctcattct cactttctga cttatagtcg ctttgttaaa
     4141 tcatatgtcg acatggtacc agatctctcg aggatccatg gctgatgtcc aactggtgga
     4201 gtctggtggc gctttggtgc agccaggtgg ctctctgcgt ttgtcctgtg ccgcttctgg
     4261 cttcccagtg aaccgctatt ccatgcgctg gtatcgccag gctccaggca aagagcgtga
     4321 gtgggtagcc ggtatgtcca gcgcgggtga tcgtagctcc tatgaagact ccgtgaaggg
     4381 ccgtttcacc atcagccgtg acgatgcccg taacacggtg tatctgcaaa tgaacagctt
     4441 gaaacctgaa gatacggccg tgtattactg taatgtgaac gtgggcttcg agtattgggg
     4501 ccaaggcacc caggtcaccg tctcctctag catggtgagc aagggcgagg aggataacat
     4561 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     4621 cgagttcgag atcgagggcg agggcgaggg ccgcccctat gagggcaccc agaccgccaa
     4681 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     4741 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     4801 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     4861 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     4921 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagacaatgg gctgggaggc
     4981 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     5041 gaagctgaag gacggtggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     5101 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctctcacaa
     5161 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgc
     5221 catggacgag ctgtacaagt aaccatgggg taaaaggaat gtctcccttg ccagtactgc
     5281 tagggttttt ctttcaaact atggaagccc attcaagctg catattacga ttttgttttt
     5341 cgcttttaga aagtggttta gatgagataa tagaaaaatt cttgatctcc gacaacgagt
     5401 acttttattt tttttgctaa tcactttact caatattagc tcgaaatcgt agaaacgtag
     5461 acgggtgcgg gataccgagt ggtgtagtta agaattttta taaaccacgt ggcccaaaaa
     5521 tatgaaccca aaacgtttat acatgagtat actttaagaa ggctataccc cttcgtgtta
     5581 gatgtagttt tagctaccca acccgagtct atgagcttga cttcagatgt agaaggcatt
     5641 aaatcgtttt gaatattaat taaaaaacga tgaaaattaa atatttaaaa gcaatcatac
     5701 gctgaaaatt tagtgctgtg gctaatcctt caacatggaa atgccataaa agtgactttg
     5761 acaaaaaaaa aagtatatac aggtagtaaa ctcatctact tcattgactt tgtttacagc
     5821 atgtggaagg aggaatattt attgctaaat cgtagtttaa cattcaataa gtaatactat
     5881 tgaaattcga caagattggc cgcatggatg aaaaagaggc attttgcttt gggagaatta
     5941 gtccaaatta gaactgaaaa aaaaaacttt acgaggcaaa aatgtcggat tgagatcgta
     6001 aaagttcgct cgtcgtcttt tgctttgtga ttgttttcat ggatacatct tgctggatat
     6061 ttaaatttta gtactatgta taagatattc tataaatgtt ttatcaccca aacctgttag
     6121 cgccttctta attctattca atctggcttt tgctctgaga ctacttcttg gactttcact
     6181 acttgttagt tatacggaat ttgtgtaatt agaagtgaaa taatcctttc tattagtaat
     6241 gcgagctccg cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaattat
     6301 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     6361 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     6421 cgttgcgccc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6481 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6541 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6601 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6661 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6721 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6781 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6841 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6901 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6961 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     7021 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     7081 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     7141 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     7201 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     7261 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     7321 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7381 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7441 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7501 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7561 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7621 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7681 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7741 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7801 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7861 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7921 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7981 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     8041 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     8101 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     8161 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     8221 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     8281 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     8341 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8401 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     8461 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     8521 gtc
//
