LOCUS Exported 7391 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION URT-type vector for Val-mI-kappa-B-alpha expression in Mammalian cells. ACCESSION . VERSION . KEYWORDS pJO428 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7391) AUTHORS Piatkov KI, Oh JH, Liu Y, Varshavsky A TITLE Calpain-generated natural protein fragments as short-lived substrates of the N-end rule pathway. JOURNAL Proc Natl Acad Sci U S A. 2014 Mar 4;111(9):E817-26. doi: 10.1073/pnas.1401639111. Epub 2014 Feb 18. PUBMED 24550490 REFERENCE 2 (bases 1 to 7391) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7391 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 629..648 /label=T7 /note="T7 promoter, forward primer" promoter 629..647 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 664..687 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 1000..1560 /codon_start=1 /gene="Mus musculus Dhfr" /product="mouse dihydrofolate reductase" /label=DHFR /translation="MVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVE GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY PGVLSEVQEEKGIKYKFEVYEKKD" CDS 1567..1593 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 2665..2688 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" promoter complement(2710..2728) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2711..2728) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2748..2765) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2754..2978 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3024..3452 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3111..3130) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3321..3342 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3461..3481) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3466..3795 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3646..3781 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3708..3727 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3862..4656 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3916..3935) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4526..4545 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4830..4951 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4867..4886) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4921..4940 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5000..5016) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5000..5016) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5013..5035) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5024..5040 /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(5048..5078) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5093..5114 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5231..5248) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5402..5990) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5482..5501) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6161..7021) /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 6784..6803 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7022..7126) /gene="bla" /label=AmpR promoter primer_bind complement(7201..7220) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt 661 atggactaca aggacgacga tgacaagggt gcacgacagg tttcccgact taatcgcctt 721 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 781 tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa 841 gcggtgccgg aaagctggct ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc 901 tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtgac ctatcccatt 961 acggtcaatc cgccgtttgt tcccacggag aatggatcca tggttcgacc attgaactgc 1021 atcgtcgccg tgtcccaaaa tatggggatt ggcaagaacg gagacctacc ctggcctccg 1081 ctcaggaacg agttcaagta cttccaaaga atgaccacaa cctcttcagt ggaaggtaaa 1141 cagaatctgg tgattatggg taggaaaacc tggttctcca ttcctgagaa gaatcgacct 1201 ttaaaggaca gaattaatat agttctcagt agagaactca aagaaccacc acgaggagct 1261 cattttcttg ccaaaagttt ggatgatgcc ttaagactta ttgaacaacc ggaattggca 1321 agtaaagtag acatggtttg gatagtcgga ggcagttctg tttaccagga agccatgaat 1381 caaccaggcc acctcagact ctttgtgaca aggatcatgc aggaatttga aagtgacacg 1441 tttttcccag aaattgattt ggggaaatat aaacttctcc cagaataccc aggcgtcctc 1501 tctgaggtcc aggaggaaaa aggcatcaag tataagtttg aagtctacga gaagaaagac 1561 ggtacctacc catacgatgt cccagattac gccgctttcc ttgcgggtag tgggtcgacc 1621 atgcagattt tcgtcaagac tttgaccggt aaaaccataa cattggaagt tgaatcttcc 1681 gataccatcg acaacgttaa gtcgaaaatt caagacaagg aaggtatccc tccagatcaa 1741 caaagattga tctttgccgg taggcagcta gaagacggta gaacgctgtc tgattacaac 1801 attcagaagg agtccacctt acatcttgtg ctaaggctcc gcggaggagt tatccgcctg 1861 cagccgcagg aggcgccgct ggccgccgag ccctggaagc agcagctcac ggaggacgga 1921 gactcgttcc tgcacttggc aatcatccac gaagagaagc cgctgaccat ggaagtcatt 1981 ggtcaggtga agggagacct ggccttcctc aacttccaga acaacctgca gcagactcca 2041 ctccacttgg ctgtgatcac caaccagcca ggaattgctg aggcacttct gaaagctggc 2101 tgtgatcctg agctccgaga ctttcgagga aatacccctc tacatcttgc ctgtgagcag 2161 ggctgcctgg ccagtgtagc agtcttgacg cagacctgca caccccagca tctccactcc 2221 gtcctgcagg ccaccaacta caatggccac acgtgtctgc acctagcctc tatccacggc 2281 tacctggcca tcgtggagca cttggtgact ttgggtgctg atgtcaacgc tcaggagccc 2341 tgcaatggcc ggacagccct ccaccttgcg gtggacctgc agaatcctga cctggtttcg 2401 ctcttgttga aatgtggggc tgatgtcaac agggtaacct accaaggcta ctccccctac 2461 cagcttacct ggggccgccc aagtacccgg atacagcagc agctgggcca gctgaccctg 2521 gaaaatctcc agatgctacc cgagagcgag gatgaggaga gctatgacac ggagtcagaa 2581 ttcacagagg atgagctgcc ctatgatgac tgtgtgtttg gaggccagcg tctgacatta 2641 atggctatgt ccatgatcga tatcgactac aaagacgatg acgacaaagg ttaatctaga 2701 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc 2761 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 2821 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 2881 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 2941 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag 3001 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 3061 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 3121 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 3181 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 3241 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 3301 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 3361 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 3421 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg 3481 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 3541 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 3601 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 3661 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 3721 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 3781 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag 3841 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc 3901 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 3961 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 4021 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg 4081 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 4141 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 4201 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 4261 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 4321 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 4381 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg 4441 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 4501 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 4561 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 4621 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga 4681 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg 4741 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg 4801 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca 4861 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 4921 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct 4981 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 5041 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 5101 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 5161 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 5221 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 5281 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 5341 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 5401 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 5461 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 5521 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 5581 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 5641 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 5701 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 5761 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 5821 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 5881 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 5941 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 6001 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 6061 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 6121 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 6181 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 6241 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 6301 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 6361 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 6421 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 6481 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 6541 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 6601 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 6661 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 6721 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 6781 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 6841 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 6901 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 6961 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 7021 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 7081 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 7141 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag 7201 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga 7261 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa 7321 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag 7381 atatacgcgt t //