LOCUS       sequence-226894-        3745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal GFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3745)
  AUTHORS   Diebold-Durand ML, Burmann F, Gruber S
  TITLE     High-Throughput Allelic Replacement Screening in Bacillus subtilis.
  JOURNAL   Methods Mol Biol. 2019;2004:49-61. doi: 10.1007/978-1-4939-9520-2_5.
  PUBMED    31147909
REFERENCE   2  (bases 1 to 3745)
  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 Mol
            Biol."; date: "2019"; volume: "2004:49-61. doi"; pages: " 
            10.1007/978-1-4939-9520-2_5"
COMMENT     Sequence Label: pJet1.2 sfGFP
FEATURES             Location/Qualifiers
     source          1..3745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     305..324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        305..323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(383..1090)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFI
                     CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANF
                     KIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFV
                     TAAGITHGMDELYK"
     primer_bind     complement(423..443)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1038..1066
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(join(1577..1583,1584..1601,1602..1607))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    1622..1643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1763..1780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1934..2522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2014..2033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2693..3484,3485..3553))
                     /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     3316..3335
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3554..3658)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3726..3744
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc
       61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc
      121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact
      181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt
      241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc
      301 ggcgtaatac gactcactat agggagagcg gccgccagat cttccggatg gctcgagttt
      361 ttcagcaaga tggtctcgct tatttgtaga gctcatccat gccatgtgta atcccagcag
      421 cagttacaaa ctcaagaagg accatgtggt cacgcttttc gttgggatct ttcgaaagga
      481 cagattgtgt cgacaggtaa tggttgtctg gtaaaaggac agggccatcg ccaattggag
      541 tattttgttg ataatggtct gctagttgaa cggaaccatc ttcaacgttg tggcgaattt
      601 tgaagttagc tttgattcca ttcttttgtt tgtctgccgt gatgtataca ttgtgtgagt
      661 taaagttgta ctcgagtttg tgtccaagaa tgtttccatc ttctttaaaa tcaataccct
      721 ttaactcgat acgattaaca agggtatcac cttcaaactt gacttcagca cgcgtcttgt
      781 aggtcccgtc atctttgaaa gatatagtgc gttcctgtac ataaccttcg ggcatggcac
      841 tcttgaaaaa gtcatgccgt ttcatgtgat ccggataacg ggaaaagcat tgaacaccat
      901 aggtcagagt agtgacaagt gttggccacg gaacaggtag ttttccagta gtgcaaataa
      961 atttaagggt gagttttccg tttgtagcat caccttcacc ctctccacgg acagaaaatt
     1021 tgtgcccatt aacatcacca tctaattcaa caagaattgg gacaactcca gtgaaaagtt
     1081 cttctccttt gcctccagat ccgcctgagc ctccgctgcc tgatccgcct ccgctcgaga
     1141 ccatctttct agaagatctc ctacaatatt ctcagctgcc atggaaaatc gatgttcttc
     1201 ttttattctc tcaagatttt caggctgtat attaaaactt atattaagaa ctatgctaac
     1261 cacctcatca ggaaccgttg taggtggcgt gggttttctt ggcaatcgac tctcatgaaa
     1321 actacgagct aaatattcaa tatgttcctc ttgaccaact ttattctgca ttttttttga
     1381 acgaggttta gagcaagctt caggaaactg agacaggaat tttattaaaa atttaaattt
     1441 tgaagaaagt tcagggttaa tagcatccat tttttgcttt gcaagttcct cagcattctt
     1501 aacaaaagac gtctcttttg acatgtttaa agtttaaacc tcctgtgtga aattattatc
     1561 cgctcataat tccacacatt atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     1621 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gccaattgct ttccagtcgg
     1681 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1741 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1801 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1861 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1921 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1981 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2041 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2101 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2161 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2221 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2281 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2341 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2401 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2461 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2521 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2581 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2641 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2701 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     2761 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     2821 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     2881 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     2941 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3001 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3061 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3121 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3181 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3241 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3301 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3361 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3421 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3481 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3541 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     3601 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3661 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     3721 ataaaaatag gcgtatcacg aggcc
//
