LOCUS Exported 6736 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pcDNA3-DN-hCUL4A-FLAG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6736) AUTHORS Jin J, Ang XL, Shirogane T, Wade Harper J TITLE Identification of substrates for F-box proteins. JOURNAL Methods Enzymol. 2005 . 399():287-309. PUBMED 16338364 REFERENCE 2 (bases 1 to 6736) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6736 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(18..37) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 209..588 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 589..792 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 743..763 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 837..856 /label=T7 /note="T7 promoter, forward primer" promoter 837..855 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 2210..2233 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" promoter complement(2263..2281) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2264..2281) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2301..2318) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2307..2531 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2577..3005 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2664..2683) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2874..2895 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3014..3034) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3019..3348 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3199..3334 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3261..3280 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3415..4209 /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(3469..3488) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4079..4098 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4383..4504 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4420..4439) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4474..4493 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4553..4569) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4553..4569) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4566..4588) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4577..4593 /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(4601..4631) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4646..4667 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4784..4801) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4955..5543) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5035..5054) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5714..6574) /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 6337..6356 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6575..6679) /gene="bla" /label=AmpR promoter ORIGIN 1 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc 61 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg 121 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt 181 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa 241 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 301 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 361 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 421 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 481 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 541 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 601 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 661 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 721 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 781 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat 841 acgactcact atagggagac ccaagcttgg taccgagctc ggatccacca tggcggacga 901 ggccccgcgg aagggcagct tctcggcgct cgtgggccgc accaacggcc tcaccaagcc 961 cgcggccctg gccgccgcgc ccgccaagcc ggggggcgcg ggcggctcca agaagctggt 1021 catcaagaac ttccgagaca gacctcggct gcccgacaac tacacgcagg acacgtggcg 1081 gaagctgcac gaggcggtgc gggccgtgca gagcagcacc tccatcaggt acaacctcga 1141 ggagctctac caggctgtgg aaaatctctg ttctcacaaa gtctccccaa tgctctacaa 1201 gcaactgcgt caggcctgtg aagaccacgt ccaggcacag atccttccgt ttagagaaga 1261 ctcactagat agtgttttat ttttaaagaa gattaacacg tgctggcagg accactgcag 1321 acaaatgatc atgatcagaa gcatcttcct gttcttggac cgcacctatg tgctgcagaa 1381 ctccacgctg ccctccatct gggatatggg attagaactg tttagaaccc atattattag 1441 tgataaaatg gttcagagta aaaccattga tggaatccta ctgctgatcg agcgcgagag 1501 gagcggcgag gccgtggacc ggagcctgtt gcggagcctc ctgggcatgc tgtctgacct 1561 gcaggtgtat aaagattcat ttgaactgaa atttttggaa gagactaatt gcttatatgc 1621 tgccgaaggc caaaggttaa tgcaggaaag agaggtccca gaatatctta accatgtaag 1681 taaacgctta gaggaagagg gagacagagt aatcacttac ttggaccaca gcacacagaa 1741 accactgatt gcttgtgtgg agaaacagct attaggagaa catttaacag caattctgca 1801 gaaagggctc gaccacttac tggatgagaa cagagtgccg gacctcgcac agatgtacca 1861 gctgttcagc cgggtgaggg gcgggcagca ggcgctgctg cagcactgga gcgagtacat 1921 caagactttt ggaacagcga tcgtaatcaa tcctgagaaa gataaagaca tggtccaaga 1981 cctgttggac ttcaaggaca aggtggacca cgtgatcgag gtctgcttcc agaagaatga 2041 gcggttcgtc aacctgatga aggagtcctt tgagacgttc atcaacaaga gacccaacaa 2101 gcctgcagaa ctgatcgcaa agcatgtgga ttcaaagtta agagcaggca acaaagaagc 2161 cacagacgag gagctggagc ggacgttgga caagatcatg atcctgttcg actacaagga 2221 tgacgatgac aagtagtcga gcatgcatct agagggccct attctatagt gtcacctaaa 2281 tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 2341 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 2401 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 2461 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 2521 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc 2581 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 2641 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 2701 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 2761 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 2821 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 2881 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 2941 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 3001 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca 3061 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca 3121 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc 3181 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc 3241 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta 3301 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga 3361 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt 3421 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat 3481 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag 3541 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac 3601 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac 3661 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc 3721 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg 3781 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag 3841 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat 3901 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag 3961 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc 4021 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg 4081 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg 4141 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag 4201 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat 4261 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc 4321 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc 4381 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 4441 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 4501 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc 4561 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 4621 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 4681 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 4741 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 4801 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 4861 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 4921 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 4981 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 5041 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 5101 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 5161 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 5221 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 5281 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 5341 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 5401 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 5461 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 5521 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 5581 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 5641 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 5701 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 5761 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 5821 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 5881 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 5941 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 6001 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 6061 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 6121 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 6181 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 6241 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 6301 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 6361 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 6421 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 6481 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 6541 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 6601 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 6661 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg 6721 gagatctccc gatccc //