LOCUS       sequence-181135-        8511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  genomic GBP-mCherry epitope N-terminal tagging at leu1+ locus in S. 
            pombe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8511)
  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 8511)
  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(N)-leu1+
FEATURES             Location/Qualifiers
     source          1..8511
                     /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(2304..2759)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2441..2462)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2653..2672
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2885..2907
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2899..2916
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2900..2916
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2926..2945
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2926..2944
                     /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             4157..4498
                     /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             4502..5209
                     /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(4647..4665)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4915..4934
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(6251..6271)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6251..6269)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6290..6306)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6290..6306)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6303..6325)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6314..6330
                     /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(6338..6344,6345..6362,6363..6368))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6383..6404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6521..6538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6692..7280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6772..6791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7451..8242,8243..8311))
                     /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     8074..8093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8312..8416)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8484..8502
                     /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 aaaaaccagc tcattgactc cgcagccatg cttttggtca agagccctcg tacacttaac
     1021 ggtgttgttt tgactgacaa cttgtttggt gacattattt cagatgaggc ttctgtcatt
     1081 cctggtagct tgggcctttt gccttctgcc tccctttccg gtgtggtagg aaaatcagaa
     1141 gaaaaggttc attgtttggt tgagcccatt cacggtagcg ctcccgatat cgctggcaag
     1201 ggcattgtta atcctgttgg tacaatttta tctgcttccc ttctccttcg ttatggtttg
     1261 aatgctccta aggaggctga agctatcgaa gccgccgtac gcaaggtctt ggatgatact
     1321 tcaattggtg gacgtggtct ttatactcgc gatttgggag gtgaggcttc taccgctgat
     1381 attactaagg ctgttgttga agaacttgaa aaaattttgt aatagggttt aatgtagaat
     1441 aaattcacat gatttgattt gacctaattt ttattgatta attgtgtcgc atatttatta
     1501 tcgttaaaaa tgcaattgac aaaagcgtaa attttaaggc tctaatgttt tttcttttgg
     1561 tatgttatag tcaagcaaag atgtattaag ttgcaaaagc atggaattga tttacgacat
     1621 ccgttcaact ttatgaccga gttgtcgctg tgtacttact ctttttttac tatgaaaagt
     1681 ttcaaacata atatcattaa ttatttcttt ttaaacgaag taacctgtaa cataaataat
     1741 aggggaaaag aagaaaaaaa ccataaaaat tcgagaacgt aacaattatt attgtaacga
     1801 tgacaacccg attctttgag agagccagag acatgacttt tgcattaccg taattggttt
     1861 ctacttatga attgtaaact tgtgtaggga atttcatcca cagctatcgc gggctgctct
     1921 ctgcatcagt gttagcacat tttatattaa tgaagatttt gctatgcaaa atacgtacat
     1981 ttctggcgca tatattttta tcaaacgaac atgactatcc tttccacttt gatggttcgc
     2041 aagtattttg atttgtatta aatgcacatc taacaagcca tttcgcccaa aatcaccaac
     2101 tggcacatgt acccaaccag ttagttcctc gacttgaact gttgtaacaa tttccaaatc
     2161 ttgaaaccct gtcccagctg atattcgcaa agtactggga gtataagatt catcaagggt
     2221 gtattgaagg tacatactca catacttaat cgatgcggtg tgaaataccg cacagatgcg
     2281 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     2341 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     2401 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     2461 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2521 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2581 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     2641 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     2701 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     2761 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     2821 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     2881 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     2941 tagggcgaat tggctgcagg 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 tcatatgatg gctgatgtcc aactggtgga gtctggtggc gctttggtgc agccaggtgg
     4201 ctctctgcgt ttgtcctgtg ccgcttctgg cttcccagtg aaccgctatt ccatgcgctg
     4261 gtatcgccag gctccaggca aagagcgtga gtgggtagcc ggtatgtcca gcgcgggtga
     4321 tcgtagctcc tatgaagact ccgtgaaggg ccgtttcacc atcagccgtg acgatgcccg
     4381 taacacggtg tatctgcaaa tgaacagctt gaaacctgaa gatacggccg tgtattactg
     4441 taatgtgaac gtgggcttcg agtattgggg ccaaggcacc caggtcaccg tctcctctag
     4501 catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
     4561 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     4621 ccgcccctat gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     4681 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     4741 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     4801 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     4861 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     4921 aatgcagaag aagacaatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     4981 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggtggcc actacgacgc
     5041 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     5101 caacatcaag ttggacatca cctctcacaa cgaggactac accatcgtgg aacagtacga
     5161 acgcgccgag ggccgccact ccaccggcgc catggacgag ctgtacaagg tcgacaagat
     5221 ctctcgagga tccatggggt aaaaggaatg tctcccttgc cagtactgct agggtttttc
     5281 tttcaaacta tggaagccca ttcaagctgc atattacgat tttgtttttc gcttttagaa
     5341 agtggtttag atgagataat agaaaaattc ttgatctccg acaacgagta cttttatttt
     5401 ttttgctaat cactttactc aatattagct cgaaatcgta gaaacgtaga cgggtgcggg
     5461 ataccgagtg gtgtagttaa gaatttttat aaaccacgtg gcccaaaaat atgaacccaa
     5521 aacgtttata catgagtata ctttaagaag gctatacccc ttcgtgttag atgtagtttt
     5581 agctacccaa cccgagtcta tgagcttgac ttcagatgta gaaggcatta aatcgttttg
     5641 aatattaatt aaaaaacgat gaaaattaaa tatttaaaag caatcatacg ctgaaaattt
     5701 agtgctgtgg ctaatccttc aacatggaaa tgccataaaa gtgactttga caaaaaaaaa
     5761 gtatatacag gtagtaaact catctacttc attgactttg tttacagcat gtggaaggag
     5821 gaatatttat tgctaaatcg tagtttaaca ttcaataagt aatactattg aaattcgaca
     5881 agattggccg catggatgaa aaagaggcat tttgctttgg gagaattagt ccaaattaga
     5941 actgaaaaaa aaaactttac gaggcaaaaa tgtcggattg agatcgtaaa agttcgctcg
     6001 tcgtcttttg ctttgtgatt gttttcatgg atacatcttg ctggatattt aaattttagt
     6061 actatgtata agatattcta taaatgtttt atcacccaaa cctgttagcg ccttcttaat
     6121 tctattcaat ctggcttttg ctctgagact acttcttgga ctttcactac ttgttagtta
     6181 tacggaattt gtgtaattag aagtgaaata atcctttcta ttagtaatgc gagctccgca
     6241 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     6301 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     6361 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     6421 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6481 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     6541 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6601 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6661 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6721 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6781 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6841 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6901 ggtatctcag ttcggtgtag gtcattcgct ccaagctggg ctgtgtgcac gaaccccccg
     6961 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     7021 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     7081 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     7141 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     7201 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     7261 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     7321 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7381 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7441 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7501 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7561 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7621 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7681 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7741 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7801 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7861 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7921 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7981 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     8041 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     8101 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     8161 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8221 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8281 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8341 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8401 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     8461 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
