LOCUS       sequence-181133-        5270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  genomic GBP-mCherry epitope C-terminal tagging in S. pombe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5270)
  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 5270)
  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: pFA6a-GBP-mCherry-kanMX6
FEATURES             Location/Qualifiers
     source          1..5270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             70..411
                     /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             415..1125
                     /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(560..578)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     828..847
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      1183..1370
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1238..1262)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1445..2801
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1445..1788
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1789..2598
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1856..1875)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2604..2801
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2905..2924)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2906..2924)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3011..3028)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3182..3770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3262..3281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3941..4732,4733..4801))
                     /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     4564..4583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4802..4906)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4974..4992
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5030..5052)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5152..5171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5252..5270
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5252..5269
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 acgcggccgc cagctgaagc ttcgtacgct gcaggtcgac ggatccccgg gttaattaac
       61 atggctgatg tccaactggt ggagtctggt ggcgctttgg tgcagccagg tggctctctg
      121 cgtttgtcct gtgccgcttc tggcttccca gtgaaccgct attccatgcg ctggtatcgc
      181 caggctccag gcaaagagcg tgagtgggta gccggtatgt ccagcgcggg tgatcgtagc
      241 tcctatgaag actccgtgaa gggccgtttc accatcagcc gtgacgatgc ccgtaacacg
      301 gtgtatctgc aaatgaacag cttgaaacct gaagatacgg ccgtgtatta ctgtaatgtg
      361 aacgtgggct tcgagtattg gggccaaggc acccaggtca ccgtctcctc tagcatggtg
      421 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
      481 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
      541 tatgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
      601 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
      661 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
      721 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
      781 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
      841 aagaagacaa tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
      901 aagggcgaga tcaagcagag gctgaagctg aaggacggtg gccactacga cgctgaggtc
      961 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     1021 aagttggaca tcacctctca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     1081 gagggccgcc actccaccgg cgccatggac gagctgtaca agtaagcggc cgctcgttcc
     1141 agattacgct gctcagtgct gaggcgcgcc acttctaaat aagcgaattt cttatgattt
     1201 atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac
     1261 tcttaggttt taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg
     1321 ctttctcagg tatagtatga ggtcgctctt attgaccaca cctctaccgg cagatccgct
     1381 agggataaca gggtaatata gatctgttta gcttgcctcg tccccgccgg gtcacccggc
     1441 cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc
     1501 atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc
     1561 catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg
     1621 cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta
     1681 gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa
     1741 aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaggaa
     1801 aagactcacg tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     1861 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     1921 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     1981 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     2041 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca
     2101 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     2161 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     2221 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     2281 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg
     2341 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     2401 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     2461 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     2521 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     2581 ctcgatgagt ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt
     2641 catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt
     2701 atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa
     2761 tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac
     2821 gccgccatcc agtttaaacg agctcgaatt catcgatgat atcagatcca ctagtggcct
     2881 atgcggccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg
     2941 ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3001 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3061 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4921 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4981 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     5041 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     5101 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     5161 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt
     5221 aatacataac cttatgtatc atacacatac gatttaggtg acactataga
//
