LOCUS Exported 3745 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION C-terminal GFP tag. ACCESSION . VERSION . KEYWORDS pJet1.2 sfGFP 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3745 /organism="synthetic DNA construct" /mol_type="other DNA" 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 (Pedelacq et al., 2006)" /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(1577..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(2693..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 //