LOCUS Exported 4519 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Artificial phase 1 exon to include a spliced T2A-LexA effector into a locus of interest through RMCE of MiMIC insertion in an intron between coding exons of a gene. ACCESSION . VERSION . KEYWORDS pBS_KS_attB2_SA(1)_T2ALexA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4519) AUTHORS Gnerer JP, Venken KJ, Dierick HA TITLE Gene-specific cell labeling using MiMIC transposons. JOURNAL Nucleic Acids Res. 2015 Apr 30;43(8):e56. doi: 10.1093/nar/gkv113. Epub 2015 Feb 20. PUBMED 25712101 REFERENCE 2 (bases 1 to 4519) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4519 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(83..100) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(254..842) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(334..353) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1013..1873) /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 1636..1655 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1874..1978) /gene="bla" /label=AmpR promoter rep_origin complement(2004..2459) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2141..2162) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2353..2372 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2586..2608 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2600..2617 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2601..2617 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 2627..2646 /label=T7 /note="T7 promoter, forward primer" promoter 2627..2645 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 2681..2750 /label=attB /bound_moiety="phage phi-C31 integrase" /note="attB site for the phi-C31 integrase (Groth et al., 2000)" CDS 2919..2972 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 2973..3578 /codon_start=1 /gene="lexA" /product="E. coli DNA-binding protein that represses genes involved in the SOS response to DNA damage" /label=LexA /note="The N-terminal DNA-binding domain recognizes the 16-bp palindromic sequence 5'-CTGTATATATATACAG-3'." /translation="MKALTARQQEVFDLIRDHISQTGMPPTRAEIAQRLGFRSPNAAEE HLKALARKGVIEIVSGASRGIRLLQEEEEGLPLVGRVAAGEPLLAQQHIEGHYQVDPSL FKPNADFLLRVSGMSMKDIGIMDGDLLAVHKTQDVRNGQVVVARIDDEVTVKRLKKQGN KVELLPENSEFKPIVVDLRQQSFTIEGLAVGVIRNGDWL" primer_bind 3516..3537 /label=LexA /note="LexA DNA binding domain, forward primer" primer_bind complement(4015..4031) /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4015..4031) /label=pBluescriptSK /note="For pBluescript vector" protein_bind 4223..4292 /label=attB /bound_moiety="phage phi-C31 integrase" /note="attB site for the phi-C31 integrase (Groth et al., 2000)" primer_bind complement(4332..4352) /label=T3 /note="T3 promoter, forward primer" promoter complement(4332..4350) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4371..4387) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4371..4387) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4384..4406) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4395..4411 /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)." promoter complement(4419..4449) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4464..4485 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." ORIGIN 1 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 61 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 121 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 181 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 241 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 301 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 361 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 421 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 481 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 541 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 601 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 661 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 721 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 781 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 841 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 901 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 961 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 1021 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 1081 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 1141 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 1201 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 1261 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 1321 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 1381 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 1441 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 1501 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 1561 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 1621 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 1681 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 1741 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 1801 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 1861 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 1921 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 1981 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt 2041 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta 2101 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc 2161 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg 2221 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact 2281 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt 2341 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc 2401 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc 2461 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct 2521 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg 2581 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact 2641 atagggcgaa ttggagctcg ctagcgatgt aggtcacggt ctcgaagccg cggtgcgggt 2701 gccagggcgt gcccttgggc tccccgggcg cgtactccac ctcacccatc tggtccatca 2761 tgatggtcga ctctagaagt cgatccaaca tggcgacttg tcccatcccc ggcatgttta 2821 aatatactaa ttattcttga actaatttta atcaaccgat ttatctctct tccgcaggtg 2881 gcggaggttc cggtggaagc ggaggtagcg gcggatccga aggacgcgga agcctgttga 2941 cgtgcggaga tgtggaagag aatccaggac cgatgaaagc gttaacggcc aggcaacaag 3001 aggtgtttga tctcatccgt gatcacatca gccagacagg tatgccgccg acgcgtgcgg 3061 aaatcgcgca gcgtttgggg ttccgttccc caaacgcggc tgaagaacat ctgaaggcgc 3121 tggcacgcaa aggcgttatt gaaattgttt ccggcgcatc acgcgggatt cgtctgttgc 3181 aggaagagga agaagggttg ccgctggtag gtcgtgtggc tgccggtgaa ccacttctgg 3241 cgcaacagca tattgaaggt cattatcagg tcgatccttc cttattcaag ccgaatgctg 3301 atttcctgct gcgcgtcagc gggatgtcga tgaaagatat cggcattatg gatggtgact 3361 tgctggcagt gcataaaact caggatgtac gtaacggtca ggtcgttgtc gcacgtattg 3421 atgacgaggt taccgttaag cgcctgaaaa aacagggcaa taaagtcgaa ctgttgccag 3481 aaaatagcga gtttaaacca attgtcgtag atcttcgtca gcagagcttc accattgaag 3541 ggctggcggt tggggttatt cgcaacggcg actggctggg atctccggcc gacgccctgg 3601 acgacttcga cctggacatg ctgccggccg acgccctgga cgacttcgac ctggacatgc 3661 tgccggccga cgccctggac gacttcgacc tggacatgct gccggggtaa ctaagtaagg 3721 atctagacat gataagatac attgatgagt ttggacaaag cttatcgata ccgtcgacta 3781 aagccaaata gaaaattatt cagttcctgg cttaagtttt taaaagtgat attatttatt 3841 tggttgtaac caaccaaaag aatgtaaata actaatacat aattatgtta gttttaagtt 3901 agcaacaaat tgattttagc tatattagct acttggttaa taaatagaat atatttattt 3961 aaagataatt gcgtttttat tgtcagggag tgagtttgct taaaaactcg tttagatcca 4021 ctagttctag agcggccgga tccggaggta gcggtggaag cggaggttcc ggaggtaagt 4081 tattgaacaa tggcatcaaa tgccttcatc atcactaccc tttagccctt aagaccccac 4141 aatgacctta cccactcaga gaaaaaagta aatatgaaag cccatttgaa cttctaagct 4201 tgtcgaccat catgatggac cagatgggtg aggtggagta cgcgcccggg gagcccaagg 4261 gcacgccctg gcacccgcac cgcggcttcg agaccgtgac ctacatcatg catggtaccc 4321 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg 4381 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cgggagcata 4441 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 4501 ctgcccgctt tccagtcgg //