LOCUS       sequence-313769-        7244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for E. coli protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7244)
  AUTHORS   Tran TH, Chan AH, Young LC, Bindu L, Neale C, Messing S, Dharmaiah 
            S, Taylor T, Denson JP, Esposito D, Nissley DV, Stephen AG, 
            McCormick F, Simanshu DK
  TITLE     KRAS interaction with RAF1 RAS-binding domain and cysteine-rich 
            domain provides insights into RAS-mediated RAF activation.
  JOURNAL   Nat Commun. 2021 Feb 19;12(1):1176. doi: 10.1038/s41467-021-21422-x.
  PUBMED    33608534
REFERENCE   2  (bases 1 to 7244)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-02-19"; volume: "12(1):1176. doi"; pages: " 
            10.1038/s41467-021-21422-x"
COMMENT     Sequence Label: pDest-521
FEATURES             Location/Qualifiers
     source          1..7244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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)."
     protein_bind    96..220
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(245..250,251..268,269..275)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             329..988
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(395..414)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1330..1635
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1514..1533
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1676..1800)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1812..1844
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1851..1868
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2034..2120
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2212..2239
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2293..2311)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2307..2354
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2391..2846
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2478..2497)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2688..2709
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2961..3752,3753..3821))
                     /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     3584..3603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3822..3914)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3995..4583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4484..4503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4737..4754
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4769..4908
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4834..4853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4994..5016
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5010..5201)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5773..6855)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6817..6836
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6856..6933)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7139..7158
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agctagatag
       61 ctagaagctt gcggccgcac agctgtatac acatcacaag tttgtacaaa aaagctgaac
      121 gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga
      181 ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat taggcacccc
      241 aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccgtc
      301 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt
      361 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg
      421 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa
      481 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc
      541 ggaattccgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg
      601 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga
      661 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct
      721 ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt
      781 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt
      841 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
      901 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta
      961 ctgcgatgag tggcagggcg gggcgtaaac gcgtggatcc ggcttactaa aagccagata
     1021 acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg atatgtatac
     1081 ccgaagtatg tcaaaaagag gtatgctatg aagcagcgta ttacagtgac agttgacagc
     1141 gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa
     1201 ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag
     1261 ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg
     1321 gctggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt
     1381 gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc
     1441 agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg
     1501 gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg
     1561 gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg
     1621 ttctggggaa tataaatgtc aggctccctt atacacagcc agtctgcagg tcgaccatag
     1681 tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca aaatctaatt
     1741 taatatattg atatttatat cattttacgt ttctcgttca gctttcttgt acaaagtggt
     1801 gatgtgcaag ccagccagaa ctcgctcctg aagacccaga ggatctcgag caccaccacc
     1861 accaccacta atgttaatta agttgggcgt tcctaggctg ataaaacaga atttgcctgg
     1921 cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag
     1981 cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa
     2041 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     2101 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     2161 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca
     2221 tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat
     2281 tcaaatatgt atccgctgag caataactag cataacccct tggggcctct aaacgggtct
     2341 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     2401 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2461 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2521 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2581 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2641 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2701 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2761 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2821 taacaaaata ttaacgttta caatttctgg cggcacgatg gcatgagatt atcaaaaagg
     2881 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2941 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3001 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3061 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3121 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3181 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3241 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3301 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3361 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3421 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3481 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3541 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3601 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3661 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3721 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3781 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatca
     3841 tgattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3901 gaaaaataaa caaataggtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3961 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4021 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4081 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4141 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4201 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4261 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4321 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4381 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4441 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4501 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4561 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4621 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4681 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4741 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4801 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4861 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4921 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4981 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5041 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     5101 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     5161 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     5221 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5281 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     5341 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     5401 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     5461 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5521 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     5581 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5641 gacaggagca cgatcatgct agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5701 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     5761 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5821 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5881 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5941 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     6001 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     6061 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     6121 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6181 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6241 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6301 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6361 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6421 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6481 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6541 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6601 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6661 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6721 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     6781 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     6841 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6901 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6961 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     7021 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     7081 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     7141 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     7201 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaa
//
