LOCUS       sequence-160022-        4875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Useful for tagging yeast proteins with C-terminal CFP tag; 
            hygromycin-B selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4875)
  AUTHORS   Hailey DW, Davis TN, Muller EG
  TITLE     Fluorescence resonance energy transfer using color variants of green
            fluorescent protein.
  JOURNAL   Methods Enzymol. 2002;351:34-49.
  PUBMED    12073355
REFERENCE   2  (bases 1 to 4875)
  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: "Methods 
            Enzymol."; date: "2002"; volume: "351"; pages: "34-49"
COMMENT     Sequence Label: pBS4
FEATURES             Location/Qualifiers
     source          1..4875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             63..773
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAH
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(90..118)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     713..733
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     gene            830..2405
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        830..1173
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1174..2202
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1174..2202
                     /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      2208..2405
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2512..2531)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2513..2531)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2618..2635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2789..3377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2869..2888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3548..4339,4340..4408))
                     /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     4171..4190
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4409..4513)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4581..4599
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4637..4659)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4759..4778
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4859..4875,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4859..4875,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggttaatta
       61 acagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
      121 atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa
      181 aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg
      241 tcactacttt gacttggggt gttcaatgct tttcaagata cccagatcat atgaaacggc
      301 atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca
      361 aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta
      421 atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat
      481 tggaatacaa ctatatttca cacaatgtat acatcactgc agacaaacaa aagaatggaa
      541 tcaaagctca tttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc
      601 attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc
      661 tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc
      721 ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac aaatagggcg
      781 cgccagatct gtttagcttg cctcgtcccc gccgggtcac ccggccagcg acatggaggc
      841 ccagaatacc ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg
      901 cccgtacatt tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg
      961 ccgcacggcg cgaagcaaaa attacggctc ctcgctgcag acctgcgagc agggaaacgc
     1021 tcccctcaca gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg
     1081 ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata
     1141 cagttctcac atcacatccg aacataaaca accatgggta aaaagcctga actcaccgcg
     1201 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     1261 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     1321 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     1381 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc
     1441 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     1501 cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
     1561 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     1621 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
     1681 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
     1741 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
     1801 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
     1861 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
     1921 ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
     1981 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
     2041 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
     2101 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
     2161 ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat aatcagtact gacaataaaa
     2221 agattcttgt tttcaagaac ttgtcatttg tatagttttt ttatattgta gttgttctat
     2281 tttaatcaaa tgttagcgtg atttatattt tttttcgcct cgacatcatc tgcccagatg
     2341 cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta
     2401 tactgctgtc gattcgatac taacgccgcc atccagtgtc gaaaacgagc tcgaattcat
     2461 cgatgatatc agatccacta gtggcctatg cggccgcgga tctgccggtc tccctatagt
     2521 gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc caacgcgcgg
     2581 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2641 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2701 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2761 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2821 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2881 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2941 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     3001 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3061 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3121 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3181 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     3241 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3301 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3361 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3421 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3481 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3541 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3601 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3661 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3721 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3781 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3841 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3901 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3961 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4021 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4081 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4141 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4201 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4261 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4321 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4381 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4441 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4501 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     4561 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     4621 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     4681 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     4741 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatggaca
     4801 tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata cacatacgat
     4861 ttaggtgaca ctata
//
