LOCUS Exported 6381 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION ManII reporter only (no hook) (RUSH system). ACCESSION . VERSION . KEYWORDS ManII-SBP-mCherry_puromycin SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6381) 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 6381) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6381 /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 1320..1433 /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 1443..2150 /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(1588..1606) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1856..1875 /label=mCherry-F /note="mCherry, forward primer" intron 2260..2489 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 2472..2491 /label=pMT2-F /note="Synthetic intron, forward primer" misc_feature 2562..3135 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2729..2746) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2956..2975 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 3168..3767 /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(3168..3187) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3664..3684 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4001..4122 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4038..4057) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(4224..4240) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4224..4240) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4237..4259) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4248..4264 /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(4272..4302) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4317..4338 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4455..4472) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4626..5214) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4706..4725) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5385..6245) /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 6008..6027 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6246..6350) /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 cgccatgaag ttaagtcgcc 961 agttcaccgt gtttggcagc gcgatcttct gcgtcgtaat cttctcactc tacctgatgc 1021 tggacagggg tcacttggac taccctcggg gcccgcgcca ggagggctcc tttccgcagg 1081 gccagctttc aatattgcaa gaaaagattg accatttgga gcgtttgctc gctgagaaca 1141 acgagattat ctcaaatatc agagactcag tcatcaacct gagcgagtct gtggaggacg 1201 gcccgcgggg gtcaccaggc aacgccagcc aaggctccat ccacctccac tcgccacagt 1261 tggccctgca ggctgacccc agagactgtt tggatcccac cggtcgccac cggaattcca 1321 tggacgagaa gaccactggt tggcgaggtg gacacgttgt tgaaggactg gctggggaac 1381 ttgaacaact tcgtgcacga ctggagcatc acccacaagg tcaacgtgaa ccacctgcag 1441 gtatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca 1501 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg 1561 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc 1621 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc 1681 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc 1741 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctacagg 1801 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg 1861 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg 1921 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg 1981 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg 2041 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg 2101 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag ggccggcctt 2161 aaggcctcga gggccaatta attaaggatc cgcggccgca tagataactg atccagtgtg 2221 ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc ctctcaaaag 2281 cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat ttgatattca 2341 cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa aagacaatct 2401 ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact tgagtgacaa 2461 tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac tgcaggtcga 2521 gcatgcatct agggcggcca attccgcccc tctccccccc ccccctctcc ctcccccccc 2581 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtga 2641 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc 2701 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat 2761 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc 2821 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt 2881 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt 2941 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag 3001 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt 3061 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga 3121 aaaacacgat gataagcttg ccacaaccca caaggagacg accttccatg accgagtaca 3181 agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg 3241 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc 3301 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt 3361 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg 3421 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg 3481 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc 3541 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc 3601 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc 3661 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc 3721 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg 3781 acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctccgaccga agccacccgg 3841 ggcggccccg ccgaccccgc acccgccccc gaggcccacc gactctagat aactgatcat 3901 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc 3961 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta 4021 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 4081 gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtcg agtgcattct 4141 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 4201 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 4261 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 4321 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 4381 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 4441 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4501 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 4561 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 4621 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4681 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 4741 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 4801 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 4861 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 4921 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 4981 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 5041 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 5101 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 5161 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 5221 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 5281 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 5341 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 5401 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 5461 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 5521 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 5581 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 5641 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 5701 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 5761 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 5821 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 5881 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 5941 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 6001 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 6061 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 6121 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 6181 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 6241 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 6301 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6361 cgaaaagtgc cacctgacgt c //