LOCUS Exported 6969 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Retroviral expression vector. Contains an altered Kozak sequence and/or upstream open reading frames to modulate expression at the level of translation.. ACCESSION . VERSION . KEYWORDS pCru5-/UAA-mEGFP-IRES-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6969) AUTHORS Ferreira JP, Overton KW, Wang CL TITLE Tuning gene expression with synthetic upstream open reading frames. JOURNAL Proc Natl Acad Sci U S A. 2013 Jul 9;110(28):11284-9. doi: 10.1073/pnas.1305590110. Epub 2013 Jun 24. PUBMED 23798422 REFERENCE 2 (bases 1 to 6969) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6969 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 32..742 /codon_start=1 /product="enhanced GFP with monomerizing A206K mutation (Zacharias et al., 2002)" /label=mEGFP /note="mammalian codon-optimized" /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF VTAAGITLGMDELYK" primer_bind complement(71..92) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(332..351) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 679..700 /label=EGFP-C /note="EGFP, forward primer" misc_feature 778..1351 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(945..962) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 1172..1191 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" regulatory 1373..1382 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1379..2089 /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(1524..1542) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1792..1811 /label=mCherry-F /note="mCherry, forward primer" LTR 2145..2738 /note="long terminal repeat from Moloney murine leukemia virus" primer_bind complement(2939..2956) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3110..3698) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3190..3209) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3872..4732) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 4495..4514 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4733..4837) /gene="bla" /label=AmpR promoter enhancer 4994..5297 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5298..5501 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" misc_feature 5740..6097 /label=MMLV Psi /note="packaging signal of Moloney murine leukemia virus (MMLV)" CDS 6162..6578 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 6508..6530 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 6546..6562 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" misc_feature 6588..6962 /label=pol region /note="Moloney murine leukemia virus (MMLV) pol region containing the splice acceptor site" primer_bind 6900..6916 /label=pBMN 5' /note="MMLV sequence, for inserts in pBMN retroviral vector" ORIGIN 1 tcgagtaact aactaataaa tgggcgaatt cagcaagggc gaggagctgt tcaccggggt 61 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg 121 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg 181 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt 241 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg 301 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga 361 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa 421 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta 481 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat 541 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg 601 ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc 661 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct 721 cggcatggac gagctgtaca agttcgaata agcatgcatc tagggcggcc aattccgccc 781 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc 841 gtttgtctat atgtgatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa 901 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat 961 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac 1021 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg 1081 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt 1141 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg 1201 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac 1261 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga 1321 cgtggttttc ctttgaaaaa cacgatgata agcttgccac aacccgggat ccgccaccat 1381 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt 1441 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg 1501 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt 1561 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc 1621 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt 1681 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg 1741 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat 1801 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc 1861 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga 1921 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa 1981 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg 2041 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaag cggccgctcg 2101 acgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc 2161 tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa 2221 ctgagaatag agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca 2281 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca 2341 gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca 2401 agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg 2461 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag 2521 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa 2581 cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca 2641 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct 2701 ctgagtgatt gactacccgt cagcgggggt ctttcatttc cgacttgtgg tctcgctgcc 2761 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtcttca catgcagcat 2821 gtatcaaaat taatttggtt ttttttctta agtatttaca ttaaatggcc atagttgcat 2881 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttggcgctct tccgcttcct 2941 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 3001 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 3061 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 3121 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 3181 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3241 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3301 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3361 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3421 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3481 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3541 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3601 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3661 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3721 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3781 caaaaaggat cttcacctag atccttttaa attaaaaatg aagtttgcgc aaatcaatct 3841 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 3901 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 3961 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 4021 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 4081 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 4141 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 4201 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 4261 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 4321 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 4381 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 4441 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata 4501 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 4561 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 4621 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 4681 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 4741 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 4801 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 4861 cagctttgct cttaggagtt tcctaataca tcccaaactc aaatatataa agcatttgac 4921 ttgttctatg ccctagttat taatagtaat caattacggg gtcattagtt catagcccat 4981 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 5041 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 5101 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 5161 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 5221 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 5281 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 5341 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 5401 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 5461 gcggtaggca tgtacggtgg gaggtctata taagcagagc tcaataaaag agcccacaac 5521 ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa 5581 taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc 5641 tgagtgattg actacccgtc agcgggggtc tttcatttgg gggctcgtcc gggatcggga 5701 gacccctgcc cagggaccac cgacccacca ccgggaggta agctggccag caacttatct 5761 gtgtctgtcc gattgtctag tgtctatgac tgattttatg cgcctgcgtc ggtactagtt 5821 agctaactag ctctgtatct ggcggacccg tggtggaact gacgagttcg gaacacccgg 5881 ccgcaaccct gggagacgtc ccagggactt cgggggccgt ttttgtggcc cgacctgagt 5941 ccaaaaatcc cgatcgtttt ggactctttg gtgcaccccc cttagaggag ggatatgtgg 6001 ttctggtagg agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg 6061 tttgggaccg aagccgcgcc gcgcgtcttg tctgctgcag catcgttctg tgttgtctct 6121 gtctgactgt gtttctgtat ttgtctgaaa atatcggccc gggccagact gttaccactc 6181 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg 6241 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg 6301 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg 6361 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag 6421 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc 6481 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat 6541 cctcccttta tccagccctc actccttctc taggcgcccc catatggcca tatgagatct 6601 tatatggggc acccccgccc cttgtaaact tccctgaccc tgacatgaca agagttacta 6661 acagcccctc tctccaagct cacttacagg ctctctactt agtccagcac gaagtctgga 6721 gacctctggc ggcagcctac caagaacaac tggaccgacc ggtggtacct cacccttacc 6781 gagtcggcga cacagtgtgg gtccgccgac accagactaa gaacctagaa cctcgctgga 6841 aaggacctta cacagtcctg ctgaccaccc ccaccgccct caaagtagac ggcatcgcag 6901 cttggataca cgccgcccac gtgaaggctg ccgaccccgg gggtggacca tcctctagac 6961 tgccggatc //