LOCUS       sequence-172773-        8540 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 8540)
  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 8540)
  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..8540
                     /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(2308..2763)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2445..2466)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2657..2676
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2895..2917
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2909..2926
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2910..2926
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2936..2955
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2936..2954
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3000..4160
                     /label=nmt1 P41 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring medium strength thiamine-repressible expression"
     CDS             4204..4545
                     /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             4549..5259
                     /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(4694..4712)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4962..4981
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(6280..6300)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6280..6298)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6319..6335)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6319..6335)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6332..6354)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6343..6359
                     /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(6367..6373,6374..6391,6392..6397))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(6550..6567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6721..7309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(7480..8271,8272..8340))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8103..8122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8341..8445)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8513..8531
                     /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 cgctggtaag
     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 tcgagaatcg taacaattat tattgtaacg
     1801 atgacaaccc gattctttga gagagccaga gacatgactt ttgcattacc gtaattggtt
     1861 tctacttatg aattgtaaac ttgtgtaggg aatttcatcc accagctatc gcgggctgct
     1921 ctctgcatca gtgttagcac attttatatt aatgaagatt ttgctatgca aaatacgtaa
     1981 atttctggcg cgatatattt ttatcaaacg aacatgacta tcctttccac tttgatggtt
     2041 cgcaagtatt ttgatttgta ttaaatgcac atctaacaag ccatttcgcc caaaatcacc
     2101 aactggcaca tgtacccaac cagttaggtt cctcgacttg aactgttgta acaatttcca
     2161 aatcttgaaa ccctgtccca gctgatattc gcaaagtact gggagtataa gattcatcaa
     2221 gggtgtattg aaggtacata ctcacatact taatcgatgc ggtgtgaaat accgcacaga
     2281 tgcgtaagga gaaaataccg catcaggaaa ttgtaaacgt taatattttg ttaaaattcg
     2341 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2401 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     2461 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2521 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     2581 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2641 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2701 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2761 cgtcgcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg cggtgcgggc
     2821 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     2881 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata
     2941 cgactcacta tagggcgaat tggctgcagg tcgatcgact ctagaggatc agaaaattat
     3001 cgccataaaa gacagaataa gtcatcagcg gttgtttcat ttcctatatt ttttttttat
     3061 ttttttattt tttaataagg gaaaatttaa cgtctaagga tacagaagat tgttagcaca
     3121 ttaaagtaat aaaggcttaa gtagtaagtg ccttagcatg ttattgtatt tcaaaggaca
     3181 taatctaaaa taataacaat atcatttctc acaagttatt caattttctt ttttttttct
     3241 aataatatca agaatgtatt atttgtttga cataagtcaa ctaatttatt taatatgctg
     3301 gattaatctt gcagacatgt aaattaacaa gttttagtca aataacgttg aagtttcaat
     3361 gaactcaaat aatttctctt tttttttata taaccataag tctaatctga tttatatttt
     3421 ccgcagggat caactgaagt tatgacattt ggattggatc acttataacc ttggtcgcca
     3481 aataatacaa aaatcagcgt tataaaacaa agaaggtttt tgttaagaaa ttaatcctct
     3541 ttcttgataa gaaagttgaa ccgaaattgc agatactgat atatgaaaat aatacccaca
     3601 attttgggaa tagcgcaagc ctcaatttaa acaataggtg aggacacatg ataatgacct
     3661 caatgattgt tagaagaaaa gagcctcatt acaaaatcga aaaatgaatg gttgggtaca
     3721 agtttccaaa acatggtaaa gtggactttg cgtatgagac gtaaatagaa aaaaacactt
     3781 gttatatgtt ttctagaatt attgttgtct ctttatggtt ggatgatgca aaatagtaat
     3841 ttcggttagt tgctgtaaaa caccacgaga caaatagata tggatattta ttaaatcagg
     3901 aaaaacgtaa ctctcggcta ctggatggtt cagtcaccca acgattactg gggagagaaa
     3961 acagggcaaa agcaaagctt aaaggaatcc gattgtcatt cggcaatgtg cagcgaaact
     4021 aaaaaccgga taatggacct gttaatcgaa acattgaaga taaaggaaga ggaatcctgg
     4081 catatcatca attgaataag ttgaattaat tatttcaatc tcattctcac tttctgactt
     4141 atagtcgctt tgttaaatca tatgtcgaca tggtaccaga tctctcgagg atccatggct
     4201 gatgtccaac tggtggagtc tggtggcgct ttggtgcagc caggtggctc tctgcgtttg
     4261 tcctgtgccg cttctggctt cccagtgaac cgctattcca tgcgctggta tcgccaggct
     4321 ccaggcaaag agcgtgagtg ggtagccggt atgtccagcg cgggtgatcg tagctcctat
     4381 gaagactccg tgaagggccg tttcaccatc agccgtgacg atgcccgtaa cacggtgtat
     4441 ctgcaaatga acagcttgaa acctgaagat acggccgtgt attactgtaa tgtgaacgtg
     4501 ggcttcgagt attggggcca aggcacccag gtcaccgtct cctctagcat ggtgagcaag
     4561 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     4621 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctatgag
     4681 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     4741 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     4801 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     4861 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     4921 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     4981 acaatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     5041 gagatcaagc agaggctgaa gctgaaggac ggtggccact acgacgctga ggtcaagacc
     5101 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     5161 gacatcacct ctcacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     5221 cgccactcca ccggcgccat ggacgagctg tacaagtaac catggggtaa aaggaatgtc
     5281 tcccttgcca gtactgctag ggtttttctt tcaaactatg gaagcccatt caagctgcat
     5341 attacgattt tgtttttcgc ttttagaaag tggtttagat gagataatag aaaaattctt
     5401 gatctccgac aacgagtact tttatttttt ttgctaatca ctttactcaa tattagctcg
     5461 aaatcgtaga aacgtagacg ggtgcgggat accgagtggt gtagttaaga atttttataa
     5521 accacgtggc ccaaaaatat gaacccaaaa cgtttataca tgagtatact ttaagaaggc
     5581 tatacccctt cgtgttagat gtagttttag ctacccaacc cgagtctatg agcttgactt
     5641 cagatgtaga aggcattaaa tcgttttgaa tattaattaa aaaacgatga aaattaaata
     5701 tttaaaagca atcatacgct gaaaatttag tgctgtggct aatccttcaa catggaaatg
     5761 ccataaaagt gactttgaca aaaaaaaaag tatatacagg tagtaaactc atctacttca
     5821 ttgactttgt ttacagcatg tggaaggagg aatatttatt gctaaatcgt agtttaacat
     5881 tcaataagta atactattga aattcgacaa gattggccgc atggatgaaa aagaggcatt
     5941 ttgctttggg agaattagtt caaattagaa ctgaaaaaaa aaactttacg aggcaaaaat
     6001 gtcggattga gatcgtaaaa gttcgctcgt cgtcttttgc tttgtgattg ttttcatgga
     6061 tacatcttgc tggatattta aattttagta ctatgtataa gatattctat aaatgtttta
     6121 tcacccaaac ctgttagcgc cttcttaatt ctattcaatc tggcttttgc tctgagacta
     6181 cttcttggac tttcactact tgttagttat acggaatttg tgtaattaga agtgaaataa
     6241 tcctttctat tagtaatgcg agctccgcag cttttgttcc ctttagtgag ggttaattgc
     6301 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     6361 tccacacaac ataggagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     6421 gtaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6481 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6541 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6601 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6661 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6721 tttccatagg ctcggccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6781 gcgaaacccg acaggactat aaagatacca ggcgttcccc cctggaagct ccctcgtgcg
     6841 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6901 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6961 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7021 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7081 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7141 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     7201 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7261 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7321 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7381 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7441 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7501 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tgcccgtcgt
     7561 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7621 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7681 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7741 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7801 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7861 aaggcgagtt acatgatccc ccatgttgtg aaaaaaagcg gttagctcct tcggtcctcc
     7921 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7981 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     8041 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     8101 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8161 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     8221 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8281 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8341 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8401 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8461 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     8521 tatcacgagg ccctttcgtc
//
