LOCUS Exported 5325 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Rab7A fused to the red fluorescent protein Ruby3 in N-ter under control of a weak L30 promoter.. ACCESSION . VERSION . KEYWORDS pBS L30 Ruby3-Rab7A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5325) AUTHORS Maika S. Deffieu, Ieva Cesonyte, Francois Delalande, Gaelle Boncompain, Cristina Dorobantu, Eli Song, Vincent Lucansky, Aurelie Hirschler, Sarah Cianferani, Tao Xu, Franck Perez, Christine Carapito, Raphael Gaudin TITLE CRISPR-based bioengineering of the Transferrin Receptor revealed a role for Rab7 in the biosynthetic secretory pathway JOURNAL Science Advances REFERENCE 2 (bases 1 to 5325) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5325 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..17 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 24..43 /label=T7 /note="T7 promoter, forward primer" promoter 24..42 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 455..464 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 461..1171 /codon_start=1 /product="monomeric red fluorescent protein derived from mRuby2, with improved brightness and photostability (Bajar et al., 2016)" /label=mRuby3 /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV AKYSNLGGGMDELYK" protein_bind 1205..1229 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(1857..1881) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(1925..1944) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 1969..2017 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" promoter complement(2233..2251) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2272..2288) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2272..2288) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2285..2307) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2296..2312 /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(2320..2350) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2365..2386 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2503..2520) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2674..3262) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2754..2773) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3433..4293) /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 4056..4075 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4294..4398) /gene="bla" /label=AmpR promoter primer_bind 4466..4484 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4522..4544) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4644..4663 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin 4730..5185 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4817..4836) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5027..5048 /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5311..8 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 5325..17 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" ORIGIN 1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattc gagctcggta 61 cccggggatc gcagaacaaa cgcccagaac cgaaggccgg agcccagacg ccgcagcagc 121 ccagcctgcg ttgtaacccg gtccccatag caaccaacta ccgcagacta ctctcgcaag 181 cgctgttctg cttgtgaccc ggaccggctt ctcttccgct tccggtccca caatcctctg 241 tcggcctaga agagctttgc attgtgggag ctccttcctt tctcgctccc cggccatctt 301 ggcggctggt gttggtgagt gagctctgcg gggtaaacga ttaggcggct cggggagctc 361 cgctagctgg tgtttgacgc tctggattat aatcctctct tctgtcctct gtgtatgcta 421 ggttgggggt tgtcccgcac ctactagagg atccgccacc atggtgtcta agggcgaaga 481 gctgatcaag gaaaatatgc gtatgaaggt ggtcatggaa ggttcggtca acggccacca 541 attcaaatgc acaggtgaag gagaaggcag accgtacgag ggagtgcaaa ccatgaggat 601 caaagtcatc gagggaggac ccctgccatt tgcctttgac attcttgcca cgtcgttcat 661 gtatggcagc cgtaccttta tcaagtaccc ggccgacatc cctgatttct ttaaacagtc 721 ctttcctgag ggttttactt gggaaagagt tacgagatac gaagatggtg gagtcgtcac 781 cgtcacgcag gacaccagcc ttgaggatgg cgagctcgtc tacaacgtca aggtcagagg 841 ggtaaacttt ccctccaatg gtcccgtgat gcagaagaag accaagggtt gggagcctaa 901 tacagagatg atgtatccag cagatggtgg tctgagagga tacactgaca tcgcactgaa 961 agttgatggt ggtggccatc tgcactgcaa cttcgtgaca acttacaggt caaaaaagac 1021 cgtcgggaac atcaagatgc ccggtgtcca tgccgttgat caccgcctgg aaaggatcga 1081 ggagagtgac aatgaaacct acgtagtgca aagagaagtg gcagttgcca aatacagcaa 1141 ccttggtggt ggcatggacg agctgtacaa gggtggttcc ggtggatctg gaggttctga 1201 tatcacaagt ttgtacaaaa aagcaggcac catgacctct aggaagaaag tgttgctgaa 1261 ggttatcatc ctgggagatt ctggagtcgg gaagacatca ctcatgaacc agtatgtgaa 1321 taagaaattc agcaatcagt acaaagccac aataggagct gactttctga ccaaggaggt 1381 gatggtggat gacaggctag tcacaatgca gatatgggac acagcaggac aggaacggtt 1441 ccagtctctc ggtgtggcct tctacagagg tgcagactgc tgcgttctgg tatttgatgt 1501 gactgccccc aacacattca aaaccctaga tagctggaga gatgagtttc tcatccaggc 1561 cagtccccga gatcctgaaa acttcccatt tgttgtgttg ggaaacaaga ttgacctcga 1621 aaacagacaa gtggccacaa agcgggcaca ggcctggtgc tacagcaaaa acaacattcc 1681 ctactttgag accagtgcca aggaggccat caacgtggag caggcgttcc agacgattgc 1741 acggaatgca cttaagcagg aaacggaggt ggagctgtac aacgaatttc ctgaacctat 1801 caaactggac aagaatgacc gggccaaggc ctcggcagaa agctgcagtt gctagaaccc 1861 agctttcttg tacaaagtgg tgatatcgta ggttgacctg cagggggaga tgggggaggc 1921 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa 1981 gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag 2041 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct 2101 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc 2161 ggggcggcaa gccctgccat agccacgggc cccgtgggtt agggacgggg tcccccatgc 2221 aagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct 2281 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 2341 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 2401 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 2461 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 2521 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 2581 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 2641 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 2701 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 2761 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 2821 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 2881 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 2941 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 3001 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 3061 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 3121 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 3181 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 3241 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 3301 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 3361 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 3421 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 3481 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 3541 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 3601 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 3661 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 3721 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 3781 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 3841 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 3901 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 3961 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 4021 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 4081 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 4141 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 4201 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 4261 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 4321 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 4381 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 4441 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg 4501 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 4561 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 4621 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat 4681 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga cgcgccctgt 4741 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 4801 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 4861 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 4921 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 4981 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 5041 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 5101 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 5161 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag 5221 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 5281 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgtt //