LOCUS       sequence-328877-        6399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for generation of N-terminal His10-tagged
            proteins in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6399)
  AUTHORS   Harel I, Chen YR, Ziv I, Singh PP, Heinzer D, Navarro Negredo P, 
            Goshtchevsky U, Wang W, Astre G, Moses E, McKay A, Machado BE, 
            Hebestreit K, Yin S, Sanchez Alvarado A, Jarosz DF, Brunet A
  TITLE     Identification of protein aggregates in the aging vertebrate brain 
            with prion-like and phase-separation properties.
  JOURNAL   Cell Rep. 2024 Jun 25;43(6):112787. doi: 
            10.1016/j.celrep.2023.112787. Epub 2024 May 28.
  PUBMED    38810650
REFERENCE   2  (bases 1 to 6399)
  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: "Cell Rep.";
            date: "2024-06-25"; volume: "43(6):112787. doi"; pages: " 
            10.1016/j.celrep.2023.112787. Epub 2024 May 28"
COMMENT     Sequence Label: pDEST17-His10-ccdB
FEATURES             Location/Qualifiers
     source          1..6399
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      115..703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     604..623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     857..874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    889..1028
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(954..973)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1114..1136
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1130..1321)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2783..2802
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2886..2905
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2886..2904
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2936..2958
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2978..3007
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3020..3031
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    3053..3177
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3202..3207,3208..3225,3226..3232)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3286..3945
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3352..3371)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4287..4592
                     /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     4471..4490
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4633..4757)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4810..4828)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4824..4871
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5237..5242,5243..5259,5260..5265))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5292..5310)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5378..5482
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5483..5551,5552..6343)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5701..5720)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
       61 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
      121 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
      181 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
      241 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
      301 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
      361 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
      421 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
      481 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
      541 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
      601 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
      661 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
      721 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
      781 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
      841 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
      901 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
      961 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     1021 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     1081 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     1141 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     1201 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     1261 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     1321 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     1381 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     1441 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     1501 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     1561 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     1621 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     1681 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     1741 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
     1801 cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct
     1861 gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg
     1921 gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct
     1981 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca
     2041 tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc
     2101 aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc
     2161 gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc
     2221 gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta
     2281 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     2341 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     2401 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     2461 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     2521 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     2581 tcgatcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     2641 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     2701 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     2761 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     2821 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     2881 gaaattaata cgactcacta tagggagacc acaacggttt ccctctagaa ataattttgt
     2941 ttaactttaa gaaggagata tacatatgtc gtactaccat caccatcacc atcaccatca
     3001 tcatcacagc agcggccata tcgaaggtcg tcatatggct agcctcgaat caacaagttt
     3061 gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
     3121 tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt cactatggcg
     3181 gccgcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa tgtgtggatt
     3241 ttgagttagg atccgtcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc
     3301 actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt
     3361 cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta
     3421 aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc
     3481 ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg
     3541 gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc
     3601 tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg
     3661 tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc
     3721 tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac
     3781 ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg
     3841 ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt
     3901 aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaaagatc tggatccggc
     3961 ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg tataagaata
     4021 tatactgata tgtatacccg aagtatgtca aaaagaggta tgctatgaag cagcgtatta
     4081 cagtgacagt tgacagcgac agctatcagt tgctcaaggc atatatgatg tcaatatctc
     4141 cggtctggta agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa
     4201 agcggaaaat caggaaggga tggctgaggt cgcccggttt attgaaatga acggctcttt
     4261 tgctgacgag aacaggggct ggtgaaatgc agtttaaggt ttacacctat aaaagagaga
     4321 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga
     4381 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc
     4441 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
     4501 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
     4561 acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata cacagccagt
     4621 ctgcaggtcg accatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt
     4681 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct
     4741 ttcttgtaca aagtggttga ttcgaggctg ctaacaaagc ccgaaaggaa gctgagttgg
     4801 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     4861 ggggtttttt gctgaaagga ggaactatat ccggatatcc acaggacggg tgtggtcgcc
     4921 atgatcgcgt agtcgatagt ggctccaagt agcgaagcga gcaggactgg gcggcggcca
     4981 aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa attgcatcaa cgcatatagc
     5041 gctagcagca cgccatagtg actggcgatg ctgtcggaat ggacgatatc ccgcaagagg
     5101 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     5161 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     5221 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg
     5281 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     5341 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5401 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5461 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5521 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5581 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5641 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5701 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     5761 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5821 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5881 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5941 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6001 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     6061 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6121 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6181 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6241 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6301 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6361 ctcatatata ctttagattg atttaaaact tcattttta
//
