LOCUS Exported 6144 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION ST reporter only ( no hook) (RUSH system). ACCESSION . VERSION . KEYWORDS ST-SBP-mCherry_puromycin SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6144) AUTHORS Boncompain G, Divoux S, Gareil N, de Forges H, Lescure A, Latreche L, Mercanti V, Jollivet F, Raposo G, Perez F TITLE Synchronization of secretory protein traffic in populations of cells. JOURNAL Nat Methods. 2012 Mar 11;9(5):493-8. doi: 10.1038/nmeth.1928. PUBMED 22406856 REFERENCE 2 (bases 1 to 6144) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6144 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1083..1196 /codon_start=1 /product="streptavidin-binding peptide" /label=SBP /note="selected from a peptide library; binds streptavidin with nanomolar affinity (Keefe et al., 2001)" /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP" CDS 1206..1913 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(1351..1369) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1619..1638 /label=mCherry-F /note="mCherry, forward primer" intron 2023..2252 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 2235..2254 /label=pMT2-F /note="Synthetic intron, forward primer" misc_feature 2325..2898 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2492..2509) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2719..2738 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 2931..3530 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(2931..2950) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3427..3447 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 3764..3885 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3801..3820) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(3987..4003) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3987..4003) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4000..4022) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4011..4027 /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(4035..4065) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4080..4101 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4218..4235) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4389..4977) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4469..4488) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5148..6008) /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 5771..5790 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6009..6113) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 901 gagctcggat cgatatctgc ggcctagcta gcgggaggcg cgccatgatt cacaccaacc 961 tgaagaaaaa gttcagctgc tgcgtcctgg tctttcttct gtttgcagtc atctgtgtgt 1021 ggaaggaaaa gaagaaaggg agttactatg attcctttaa attgcaaacc aaggggaatt 1081 ccatggacga gaagaccact ggttggcgag gtggacacgt tgttgaagga ctggctgggg 1141 aacttgaaca acttcgtgca cgactggagc atcacccaca aggtcaacgt gaaccacctg 1201 caggtatggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct 1261 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg 1321 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc 1381 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga 1441 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg 1501 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctac 1561 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc 1621 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg 1681 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg 1741 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca 1801 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt 1861 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagggccggc 1921 cttaaggcct cgagggccaa ttaattaagg atccgcggcc gcatagataa ctgatccagt 1981 gtgctggaat taattcgctg tctgcgaggg ccagctgttg gggtgagtac tccctctcaa 2041 aagcgggcat gacttctgcg ctaagattgt cagtttccaa aaacgaggag gatttgatat 2101 tcacctggcc cgcggtgatg cctttgaggg tggccgcgtc catctggtca gaaaagacaa 2161 tctttttgtt gtcaagcttg aggtgtggca ggcttgagat ctggccatac acttgagtga 2221 caatgacatc cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcaggt 2281 cgagcatgca tctagggcgg ccaattccgc ccctctcccc cccccccctc tccctccccc 2341 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg 2401 tgattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc 2461 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg 2521 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg 2581 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca 2641 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata 2701 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc 2761 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt 2821 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt 2881 tgaaaaacac gatgataagc ttgccacaac ccacaaggag acgaccttcc atgaccgagt 2941 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta cgcaccctcg 3001 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac cgccacatcg 3061 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg 3121 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag 3181 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg 3241 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt 3301 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg 3361 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg 3421 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg 3481 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc 3541 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tggctccgac cgaagccacc 3601 cggggcggcc ccgccgaccc cgcacccgcc cccgaggccc accgactcta gataactgat 3661 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct 3721 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc 3781 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 3841 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg tcgagtgcat 3901 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc 3961 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 4021 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 4081 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 4141 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 4201 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 4261 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 4321 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 4381 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 4441 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 4501 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 4561 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 4621 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 4681 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 4741 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 4801 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 4861 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 4921 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 4981 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 5041 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 5101 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 5161 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 5221 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 5281 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 5341 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 5401 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 5461 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 5521 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 5581 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 5641 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 5701 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 5761 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 5821 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 5881 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 5941 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 6001 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 6061 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 6121 ccccgaaaag tgccacctga cgtc //