LOCUS       sequence-174919-        8094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  genomic GBP-mCherry epitope N-terminal tagging at lys1+ locus in S. 
            pombe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8094)
  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 8094)
  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: pUC119-Padh81-GBP-mCherry(N)-hphMX6-lys1*
FEATURES             Location/Qualifiers
     source          1..8094
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        244..979
                     /label=adh1 promoter
                     /note="promoter for the Schizosaccharomyces pombe adh1 gene
                     encoding alcohol dehydrogenase"
     CDS             996..1337
                     /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             1341..2048
                     /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(1486..1504)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1754..1773
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      2147..2334
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2202..2226)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2409..3984
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        2409..2752
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2753..3781
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2753..3781
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      3787..3984
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(5223..5240)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5223..5239)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5232..5254)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5452..5907
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5539..5558)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5749..5770
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5924..5943)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6043..6065
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6103..6121)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6189..6293
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6294..6362,6363..7154)
                     /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(6512..6531)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7325..7913
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7814..7833
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8067..8084
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 catgccctac aacaactaag aaaatggcta tcatgcggaa gctggtgaga agaacagcat
      301 cgggacaagg gaaggaagaa caaagacaaa gaaaacaaaa gaaagcaatt gaaaacaaaa
      361 caaaacaatt ttcattcctt ctcttatcat tccttttctt ttcttttctc tcattcaacg
      421 cactccatcg tatccgtatt cctcttattt tttctctttc tctatatcca tttctttctc
      481 tctaggtgtg tcctctctct ctcttcaatt tctctactcc gcattccaac gcatccttcc
      541 cccaacctcc catttcctcc ttacggcccg atagcgatcg tctttccctc gctatcactc
      601 gctaccggcc cctcctctgc accgtaacct cctacgtatt taccatatca taaagttttt
      661 tccgacgctt atcgctgacc ccctgtcgcc ctcctattgg cttccggatt atcttcttgt
      721 ccataaggtg atccatgctt cctgaagatt cccgaaatgt gtccactttg gcggggaatc
      781 attccatcca cttctttctc tctcgctttc ctcattcggc gctccccttc cgcgtctcat
      841 tggtcttccg ctccgttttt gctttgccga tgttacttgg ggagaggtgc gataatcctt
      901 tcgcaaaaac tcggtttgac gcctcccatg gatgtgggtg gtggacaggt gccttcgctt
      961 ttctttaagc aagagaattg catatgatgg ctgatgtcca actggtggag tctggtggcg
     1021 ctttggtgca gccaggtggc tctctgcgtt tgtcctgtgc cgcttctggc ttcccagtga
     1081 accgctattc catgcgctgg tatcgccagg ctccaggcaa agagcgtgag tgggtagccg
     1141 gtatgtccag cgcgggtgat cgtagctcct atgaagactc cgtgaagggc cgtttcacca
     1201 tcagccgtga cgatgcccgt aacacggtgt atctgcaaat gaacagcttg aaacctgaag
     1261 atacggccgt gtattactgt aatgtgaacg tgggcttcga gtattggggc caaggcaccc
     1321 aggtcaccgt ctcctctagc atggtgagca agggcgagga ggataacatg gccatcatca
     1381 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     1441 tcgagggcga gggcgagggc cgcccctatg agggcaccca gaccgccaag ctgaaggtga
     1501 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     1561 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     1621 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     1681 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     1741 tcccctccga cggccccgta atgcagaaga agacaatggg ctgggaggcc tcctccgagc
     1801 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     1861 acggtggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     1921 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctctcacaac gaggactaca
     1981 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcgcc atggacgagc
     2041 tgtacaaggg taccctcgag ctagcgtcga ctctagagga tcctatccag cggccgctcg
     2101 ttccagatta cgctgctcag tgctgaggcg cgccacttct aaataagcga atttcttatg
     2161 atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag
     2221 tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag
     2281 gttgctttct caggtatagt atgaggtcgc tcttattgac cacacctcta ccggcagatc
     2341 cgctagggat aacagggtaa tatagatctg tttagcttgc ctcgtccccg ccgggtcacc
     2401 cggccagcga catggaggcc cagaataccc tccttgacag tcttgacgtg cgcagctcag
     2461 gggcatgatg tgactgtcgc ccgtacattt agcccataca tccccatgta taatcatttg
     2521 catccataca ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcgga
     2581 cctgcgagca gggaaacgct cccctcacag acgcgttgaa ttgtccccac gccgcgcccc
     2641 tgtagagaaa tataaaaggt taggatttgc cactgaggtt cttctttcat atacttcctt
     2701 ttaaaatctt gctaggatac agttctcaca tcacatccga acataaacaa ccatgggtaa
     2761 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     2821 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     2881 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     2941 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     3001 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     3061 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     3121 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     3181 tcaatacact acatggcgtg atttcatctg cgcgattgct gatccccatg tgtatcactg
     3241 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     3301 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     3361 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     3421 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     3481 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     3541 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     3601 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     3661 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     3721 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     3781 atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt atagtttttt
     3841 tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt ttttcgcctc
     3901 gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt
     3961 atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca tccagtgtcg
     4021 aaaacgagct ctcggaatac gcgatcgtat gtatcgttct ggtgacttgg gccgctatct
     4081 cccaactggt aatgtagaat gtagcggtcg tgccgacgat caaattaaaa ttcgtggttt
     4141 ccgaattgag ttaggtgaaa ttaataccca tctttctcgt catcctaatg tccgtgaaaa
     4201 tattaccctt gttcgacgtg ataaggatga agaacccacg ctagttgcat acattgtacc
     4261 tcaaggttta aataaagatg actttgatag cgctactgag tcggaagata ttgtcgtcaa
     4321 tggtttgaag aagtacagga agcttataca tgatatacgg gaatatctta agactaaact
     4381 tcctagttat gctattcctt ctgtaattgt tccccttcat aaaatgcctc tcaatcctaa
     4441 tggaaagata gataaacctg ctctgccttt cccagacaca tcacaacttg ctgccgcatc
     4501 tcgttcacat tctaaacatg gagttgatga aactcttaca gccactgaaa gggatatacg
     4561 tgatatctgg ttgcgtatca ttccacacgc tactgatgtc aacaaaaaag caagtttctt
     4621 tgatattgga ggacattcta ttttagctac tcgcttaatt tttgaacttc gcaaaaaatt
     4681 tgctgtgaat gtacctttag gtttagtttt cagtgaacct actattgaag gtttggccaa
     4741 agagattgaa agaatgaaat cgggtgagat gattagcgtg atggatatag gcaaagaaga
     4801 gactagagaa cctgaaattg aatatggtaa agatgcactt gatttagtag atttaattcc
     4861 taaagaattt ccaacctcta aagacttggg tattgatgag ccaaaaaccg ttttcttaac
     4921 tggtgctaac ggctaccttg gtgtctttat attaagagac cttatgacca gaagctctaa
     4981 cttgaaagtc attgccttag tccgcgcctc atcggaagaa catggtctaa aacgtcttaa
     5041 agacagttgt accgcgtatg gtgtatggga tgaatcatgg gcccaaaaaa tttctgttgt
     5101 caatggtgat cttgctctcg aaaactgggg tatagaagaa aggaaatgga acaagcttac
     5161 tgaagtcgtc gattatgtta ttcataatgg tgcccttgta cattgggttt atccatgaat
     5221 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     5281 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     5341 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     5401 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     5461 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5521 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5581 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5641 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     5701 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5761 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     5821 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5881 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     5941 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     6001 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     6061 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     6121 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     6181 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6241 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6301 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6361 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6421 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6481 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6541 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6601 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6661 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6721 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6781 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6841 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6901 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6961 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     7021 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     7081 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7141 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7201 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7261 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7321 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7381 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7441 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     7501 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7561 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7621 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7681 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7741 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7801 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7861 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7921 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7981 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     8041 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
