LOCUS       sequence-328879-        7003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for generation of N-terminal His10-tagged
            and C-terminal SNAP-tagged proteins in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7003)
  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 7003)
  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-SNAP
FEATURES             Location/Qualifiers
     source          1..7003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     73..92
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        73..91
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             123..145
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             165..194
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             207..218
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    240..364
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(389..394,395..412,413..419)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             473..1132
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(539..558)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1474..1779
                     /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     1658..1677
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1820..1944)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1946..1969
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1988..2533
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(2601..2619)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2615..2662
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3028..3033,3034..3050,3051..3056))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3083..3101)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3169..3273
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3274..3342,3343..4134)
                     /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(3492..3511)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4305..4893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4794..4813
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5047..5064
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5079..5218
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5144..5163)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5304..5326
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5320..5511)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6973..6992
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
       61 atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc tctagaaata
      121 attttgttta actttaagaa ggagatatac atatgtcgta ctaccatcac catcaccatc
      181 accatcatca tcacagcagc ggccatatcg aaggtcgtca tatggctagc ctcgaatcaa
      241 caagtttgta caaaaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa
      301 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
      361 tatggcggcc gcattaggca ccccaggctt tacactttat gcttccggct cgtataatgt
      421 gtggattttg agttaggatc cgtcgagatt ttcaggagct aaggaagcta aaatggagaa
      481 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
      541 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc
      601 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct
      661 tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt
      721 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc
      781 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga
      841 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt
      901 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat
      961 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt
     1021 gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag
     1081 aatgcttaat gaattacaac agtactgcga tgagtggcag ggcggggcgt aaagatctgg
     1141 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     1201 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     1261 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     1321 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     1381 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     1441 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     1501 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     1561 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     1621 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1681 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     1741 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     1801 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt
     1861 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     1921 ttcagctttc ttgtacaaag tggttgacta caaggatgac gatgacaagc ttaccggtga
     1981 gttcgctatg gacaaagatt gcgaaatgaa acgtaccacc ctggatagcc cgctgggcaa
     2041 actggaactg agcggctgcg aacagggcct gcatgaaatt aaactgctgg gtaaaggcac
     2101 cagcgcggcc gatgcggttg aagttccggc cccggccgcc gtgctgggtg gtccggaacc
     2161 gctgatgcag gcgaccgcgt ggctgaacgc gtattttcat cagccggaag cgattgaaga
     2221 atttccggtt ccggcgctgc atcatccggt gtttcagcag gagagcttta cccgtcaggt
     2281 gctgtggaaa ctgctgaaag tggttaaatt tggcgaagtg attagctatc agcagctggc
     2341 ggccctggcg ggtaatccgg cggccaccgc cgccgttaaa accgcgctga gcggtaaccc
     2401 ggtgccgatt ctgattccgt gccatcgtgt ggttagctct agcggtgcgg ttggcggtta
     2461 tgaaggtggt ctggcggtga aagagtggct gctggcccat gaaggtcatc gtctgggtaa
     2521 accgggtctg ggacctgcag gctgataagg attcgaggct gctaacaaag cccgaaagga
     2581 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     2641 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg
     2701 gtgtggtcgc catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg
     2761 ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca
     2821 acgcatatag cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat
     2881 cccgcaagag gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga
     2941 cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt
     3001 agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat
     3061 gagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3121 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3181 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3241 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3301 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3361 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3421 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3481 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     3541 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     3601 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3661 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3721 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3781 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     3841 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3901 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3961 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     4021 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4081 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4141 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4201 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4261 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4321 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4381 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4441 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4501 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4561 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4621 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4681 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4741 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4801 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4861 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4921 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4981 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     5041 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     5101 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     5161 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     5221 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5281 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5341 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     5401 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     5461 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     5521 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     5581 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     5641 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     5701 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     5761 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     5821 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     5881 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     5941 ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga
     6001 gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt
     6061 ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa
     6121 tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg
     6181 cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg
     6241 ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa
     6301 gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc
     6361 tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc
     6421 ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg
     6481 tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     6541 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     6601 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     6661 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     6721 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgatcgacg
     6781 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     6841 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     6901 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     6961 atcttcccca tcggtgatgt cggcgatata ggcgccagca acc
//
