LOCUS       sequence-98885-e        8587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To construct N-terminal GFP fusions under control of a Zn2+ 
            inducible promoter, integrates via double crossover at bgaA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8587)
  AUTHORS   Eberhardt A, Wu LJ, Errington J, Vollmer W, Veening JW
  TITLE     Cellular localization of choline-utilization proteins in 
            Streptococcus pneumoniae using novel fluorescent reporter systems.
  JOURNAL   Mol Microbiol. 2009 Oct;74(2):395-408. doi: 
            10.1111/j.1365-2958.2009.06872.x. Epub 2009 Sep 8.
  PUBMED    19737355
REFERENCE   2  (bases 1 to 8587)
  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"; doi: 
            "10.1111/j.1365-2958.2009.06872.x"; journalName: "Mol Microbiol"; 
            date: "2009-10"; volume: "74"; issue: "2"; pages: "395-408"
COMMENT     Sequence Label: pJWV25
FEATURES             Location/Qualifiers
     source          1..8587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             816..1526
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(843..871)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1466..1486
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2552..2574)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2660..2800
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2715..2734
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2815..2832)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2986..3574)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3066..3085)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3745..4536,4537..4605))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4368..4387
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4606..4710)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4778..4796
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctagcgctat atgcgttgat gcaatttcta tgcgcacccg ttctcggagc actgtccgac
       61 cgctttggcc gccgcccagt cctgctcgct tcgctacttg gagccactat cgactacgcg
      121 atcatggcga ccacacccgt cctgtggatc tatcgatgca tgcatcggta cctgcgaatt
      181 ctagatggct tttttggttt tgctgatacg gcggtcaatg ttagtcatat ggacacttaa
      241 ggcaaattgt tcagaactga ataaagctga cgttttgctt ctatcctttc tttgagtttt
      301 agtggataat gataatgaac aaggtgttca taaatctatt ataacaaagg aatgagaaat
      361 atgaaggcaa aatatgctgt ttgggtggct tttttcttaa atttgactta tgccattgtt
      421 gagtttattg caggtggagt atttggttct agcgctgttc ttgctgactc tgtgcatgac
      481 ttgggagatg cgattgcaat tggaatatca gcttttctag aaacaatctc caatcgtgaa
      541 gaagacaatc agtacacctt gggctataag cggtttagcc tgctaggagc cttagtaaca
      601 gctgtgattc tcgtaacggg ctctgttcta gtcattttgg aaaatgtcac gaagattttg
      661 catccgcaac cagtcaatga tgaggggatt ctctggttag gaattattgc gattactatc
      721 aatctgttaa cgagtctggt ggttggtaag ggaaagacaa agaatgagtc tattctgagt
      781 ctgcggatct taaggaggca aatatgaaac atcttagcaa aggagaagaa cttttcactg
      841 gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca
      901 gtggagaggg tgaaggtgat gctacatacg gaaagcttac ccttaaattt atttgcacta
      961 ctggaaaact acctgttcca tggccaacac ttgtcactac tttgacctat ggtgttcaat
     1021 gcttttcccg ttatccggat catatgaaac ggcatgactt tttcaagagt gccatgcccg
     1081 aaggttatgt acaggaacgc actatatctt tcaaagatga cgggaactac aagacgcgtg
     1141 ctgaagtcaa gtttgaaggt gatacccttg ttaatcgtat cgagttaaaa ggtattgatt
     1201 ttaaagaaga tggaaacatt ctcggacaca aactcgagta caactataac tcacacaatg
     1261 tatacatcac ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa attcgccaca
     1321 acattgaaga tggatccgtt caactagcag accattatca acaaaatact ccaattggcg
     1381 atggccctgt ccttttacca gacaaccatt acctgtcgac acaatctgcc ctttcgaaag
     1441 atcccaacga aaagcgtgac cacatggtcc ttcttgagtt tgtaactgct gctgggatta
     1501 cacatggcat ggatgagctc tacaaatcta gaggatctgg tggagaagct gcagctaaag
     1561 ctggaactag tggagcggcc gcataataag ctgagcgccg gtcgctacca ttaccagttg
     1621 gtctggtgtc aaaaataata ataaccgggc aggccatgtc tgcccgtatt tcgcgtaagg
     1681 aaatccatta tgtactattt ctggtgatga aatcaacgta acatttaaag ctgtcaaagc
     1741 caaagtcatg agatggcgta tggagcgtaa agctgacaag agcggtgttg cgatgattga
     1801 gatgaccttc cttgcaccaa gtgaattgcc tcaagaaagc actcaatcaa agattcttgt
     1861 agatggaaaa gaacttgctg atttcgctga aaatcgtcaa gactatcaaa ttacctataa
     1921 aggtcaacgg ccaaaagtct cagttgaaga aaacaatcaa gtagcttcaa ctgtggtaga
     1981 tagtggagaa gatagccttc cagtacttgt tcgcctcgtt tcagaaagtg gaaaacaagt
     2041 caaggaatac cgtatccagt tgactaagga aaaaccagtt tctgctgtac aagaagatct
     2101 tccaaaactc gaatttgttg aaaaagattt ggcctacaag acagttgaga aaaaagattc
     2161 aacactgtat ctaggtgaaa ctcgtgtaga acaagaagga aaagttggaa aagaacgtat
     2221 ctttacagtg attaatcctg atggaagtaa ggaagaaaaa ctccgtgaag tggtagaagt
     2281 tccgacagac cgcatcgtct tggttggaac caaaccagta gctcaagaag ctaaaaaacc
     2341 acaagtgtca gaaaaagcag atacaaaacc aattgattca agtgaagctg atcaaactaa
     2401 taaagcccag ttaccaaata caggtagtgc ggcaagccaa gcagcagtag cagcaggttt
     2461 agctcttcta ggtttgagtg caggattagt agttactaaa ggtaaaaaag aagactagct
     2521 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     2581 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     2641 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
     2701 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
     2761 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     2821 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2881 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2941 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3001 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3061 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3121 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3181 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3241 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3301 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3361 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3421 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3481 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3541 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3601 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3661 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3721 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3781 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3841 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3901 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3961 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4021 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg
     4081 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4141 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4201 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4261 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4321 atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc
     4381 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4441 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4501 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4561 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4621 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4681 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4741 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     4801 tcgtcttcaa gaattaattc cttcttaacg ccccaagttc atcaccaatg acatcaactc
     4861 acatgaacta catgatgaac ccagttatca tggttttgga taagattttt gaaaaattct
     4921 tcccaggcct tgataaatat gactttgatg ctgctaaatt gaacaagaaa atcggtttct
     4981 ggggatctaa attcttcatc ggtttcatcc ttggtatcgt tatcggtatt atgggaactc
     5041 cacatccaat tgcaggtgtt gcagatgcag ataaatggcg tcttgttatc aaaggatggt
     5101 tgtctcttgg tttgactgcc ggtgtatctt tggaactctt ctcacttatc ggttcatggt
     5161 tcatcgcagc cgtagaacca ctatcacaag gtattacaaa cgttgctact aaacgtcttc
     5221 aaggacgtaa attcaatatc ggtcttgact ggccattcat cgctggtcgt gctgaaatct
     5281 gggcttgtgc caacgtactt gcaccaatca tgttgattga agcagtgctt ctttcaaaag
     5341 ttggaaatgg tatcttgcca cttgcaggta tcatcgctat gggtgttact ccagctctct
     5401 tggttgtaac tcgtggtaaa ttgctccgta tgattatctt cggaacactc ttgttgccac
     5461 tcttccttct ttcaggtaca cttattgcac catttgcaac agaacttgct aaaggtgtag
     5521 gtgccttccc agaaggtgtg agccaaactc aattgattac tcactctact cttgaaggac
     5581 caatcgaaaa acttcttggt tggacaattg gtaacactac aactggtgat atcaaagcaa
     5641 tccttggtgc agtagccttc cttgtattct atatcggtat ctttgcttgg tacagaaaac
     5701 aaatgatcaa acgtaacgaa gagtacgcag caaaagcaaa ataatgcgct ccgctagctt
     5761 tacagacaaa gaactatcct taatggaagc actagcaaaa ggtatcaatg aagcaagaaa
     5821 tattgaaggc tagtcagtaa aattcagata aacaaaaaga gccgataaga tgagagtttc
     5881 atttctcaaa ttatcggctc tgcgtcttgg cgtctggctc tttgattgtt attatattca
     5941 ttttttgaac attaattaaa gtttcgttct tacatttggg gcagtataaa gggaaatttt
     6001 taagtatagt atccactcgt attcgtagtc ttgttttatt tccacaaata ggacacaata
     6061 tccacttgta gtttataata acaatctcct cctttccact ttaattcaaa tctatattaa
     6121 agaatatttc atcttattta ataagaaacc atatttatat aacaacataa aacgcactaa
     6181 gttattttat tgaacatata tcttacttta tctatctgac tatttagacg acgggtctgg
     6241 caaacaggtt cgccagtggt aacctgatat ccttttagct ctgctaaaca aacactaagc
     6301 ccatttgtaa aaaaagttaa atcattgcga taatcttgaa tacatcgagc aggaatttct
     6361 ccaataataa tgacctcatt atttttcagt tgagtattta cgatatttgc acaatatttg
     6421 ggagcatcgt tatatgcccg tgaaagatat tcctgtggtg cataaacttt aaaactaaga
     6481 tatggctcta acaattctgt tccagctttt ctaaaggctt gctccaatac aataggagca
     6541 agcatccgaa aatctgctgg ggtactaaca gggctatagt ataagccata cttaaaacag
     6601 attttacagt ccgtcacatt ccaaccatac aatccttgtt cacaaccata gcgtatccct
     6661 tccataactg cattttgaaa tgattgattt aagtatccaa gagaaaccga gctctcatac
     6721 tgcattccac ttcccaacgg aagcggtgat acagataaac caatggaagc ccagaaagga
     6781 tttggcggca cttcgatgtg aatggtatat tctgcatttt ttaacggtct ctccatataa
     6841 atgactgtag gctcttttag ttctatctcc acatgatact tttcttgcaa cagtgcacta
     6901 atcacttcca tttgtacttt ccctaagaaa gaaagtataa tttcatgtgt cgtagaatcc
     6961 acgtaatatc gtagaagcgg atcactatct gagatttcca aaagggcatc aagcaacatt
     7021 tctctctgtt caggtttact cggttcaaca gttgtttgta gtagagggtg cggattttca
     7081 atcttttttc tctgtggcaa tagttttgta tctccaagaa cactatttaa cttcaaaaac
     7141 tcattttgca aaataacaat ttctccagaa taagctctat caatcttaca taattcacca
     7201 tttattgaag tatacatttc tgtaactttt attttttctt tttctgatac tctaaccgaa
     7261 tctcgtaaat gtagtactcc actataaagg cgtatatatg caagacgttg tctttttttt
     7321 gtatattcaa ttttgaaaac atttccgcaa agttcagacg gacctcgatg tgttgatgaa
     7381 taaaatttat tagtaataac ttctataagg ttatcaatcc ctatattact ttttgcactt
     7441 ccatgataaa gagggaacag agaacaattc tgaaatctta tgctttcctc ttgttcgagt
     7501 tccaatgctt ctaatgattt accggacata tatttctcta aaaggtcatc gtttccctct
     7561 attaccgtat cccattgttc agattcggta aagttcgtca cacacacatt aggatacagt
     7621 tctaccttct gtttgattac aatttcggca gaaagtttct ctttaatatc ctgataaacc
     7681 gttgataaat caattccatt ttggtcaatc ttattgataa aaaagattgt gggaatcccc
     7741 attttcctaa gtgcatgaaa taatatacga gtttgtgctt gtacgccatc ttttgcagaa
     7801 atcagtagaa ttgccccatc taaaactgat aatgaacgat atacttctgc taagaaatcc
     7861 atatgtcctg gcgtgtctat gatgttcacc ttcgtatttt cccactgaaa agaggttatt
     7921 cctgtctgaa ttgtaattcc tctctgacgt tctaaaagcg tattatccgt cctcgttgta
     7981 cctttgtcca cgcttcctaa ttctgtaatc gctccactgt tatataataa gctttctgtt
     8041 aaggtagttt ttcctgcatc aacatgagct aaaactccaa tattaataat tttcatgtga
     8101 ttttcctcca ttcaaaagcc caaaagggca taaaaatccc agtgataaat actcttatca
     8161 ctgggatttt tatgcataac cataggcata caaagcatac agatattctc cggatacttt
     8221 agaatcacat gataaaggta ttcttaaact gggtacaaaa aactaagccc tcctaaaaaa
     8281 ggacatccaa ttatttgttc ccgctatcaa attgacagtt tatttaagaa taccttgccg
     8341 catatttatt aactcctttt aaatagatac ttaaattata gcacgtaaga gcatatttgt
     8401 aaaggaatct ccaatttttt atcaaaaaga gtacatgatt acaaagtatc tgtaatcatg
     8461 taccaatatt tgttatttta caatcttcca attatcttgt ttttctaata tcaactcaaa
     8521 ttgcgagatt tgggttgcct ttgtttcttg atctaaatat ttcacagcga cattcacgat
     8581 tacttgg
//
