LOCUS       sequence-272126-        8204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the full-length Beclin-1 protein (a.a. 1-450) with a 
            N-terminal hexa-Histidine and solubility tag (i.e., Trigger Factor).
            Three mutations are added to the CC and ECD/BARA interface..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8204)
  AUTHORS   Ranaghan MJ, Durney MA, Mesleh MF, McCarren PR, Garvie CW, Daniels 
            DS, Carey KL, Skepner AP, Levine B, Perez JR
  TITLE     The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and 
            BARA Domains To Form a Homodimer with Submicromolar Affinity.
  JOURNAL   Biochemistry. 2017 Dec 26;56(51):6639-6651. doi: 
            10.1021/acs.biochem.7b00936. Epub 2017 Dec 14.
  PUBMED    29185708
REFERENCE   2  (bases 1 to 8204)
  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"; doi: 
            "10.1021/acs.biochem.7b00936"; journalName: "Biochemistry"; date: 
            "2017-12-26- 26"; volume: "56"; issue: "51"; pages: "6639-6651"
COMMENT     Sequence Label: BPV00914 pET21GG2 H6-TF-BECN (1-450) [VA-MA-LA]
FEATURES             Location/Qualifiers
     source          1..8204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             95..112
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1433..1450
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1457..1477
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      2580..2589
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2852..2875
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2876..2920
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2936..2953
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3006..3024)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3020..3067
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3104..3559
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3191..3210)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3401..3422
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3585..3689
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3690..3758,3759..4550)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3908..3927)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4721..5309
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5210..5229
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5463..5480
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5495..5634
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5560..5579)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5720..5742
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5736..5927)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6736..7818)
                     /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     7780..7799
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7819..7896)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8102..8121
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggtcatcat catcaccatc atggtagcat
      121 gcaggttagc gttgaaacca cccagggtct gggtcgtcgt gttaccatta ccattgcagc
      181 agatagcatt gaaaccgcag ttaaaagcga actggttaac gttgcaaaaa aagtgcgcat
      241 tgatggtttt cgcaaaggta aagttccgat gaatattgtt gcacagcgtt atggtgcaag
      301 cgttcgtcag gatgttctgg gtgatctgat gagccgtaac tttattgatg ccatcatcaa
      361 agaaaaaatc aatccggcag gcgcaccgac ctatgttccg ggtgaatata aactgggtga
      421 agattttacc tacagcgtgg aatttgaagt ttatccggaa gttgaactgc agggcctgga
      481 agcaattgaa gttgaaaaac cgattgttga agttaccgat gcagatgttg atggtatgct
      541 ggataccctg cgtaaacagc aggcaacctg gaaagaaaaa gatggtgcag ttgaagccga
      601 agatcgtgtg accattgatt tcaccggtag cgttgatggt gaagaatttg agggtggtaa
      661 agcaagcgat tttgttctgg caatgggtca gggtcgtatg attccgggtt ttgaagatgg
      721 tattaaaggt cataaagccg gtgaggaatt taccattgat gttacctttc cggaagaata
      781 tcatgccgaa aatctgaaag gcaaagcagc caaatttgcc atcaatctga aaaaagtgga
      841 agaacgtgaa ctgccggaac tgaccgcaga atttatcaaa cgttttggtg tggaagatgg
      901 cagcgttgaa ggtctgcgtg ccgaagttcg caaaaatatg gaacgcgaac tgaaaagcgc
      961 aattcgtaat cgtgttaaaa gccaggccat tgaaggcctg gttaaagcaa atgatattga
     1021 tgttcctgca gccctgattg atagcgaaat tgatgtgctg cgtcgtcagg cagcccagcg
     1081 ctttggtggt aatgaaaaac aggcactgga actgcctcgt gaactgtttg aagaacaggc
     1141 aaaacgtcgt gttgttgttg gtctgctgct gggcgaagtt attcgtacca atgaactgaa
     1201 agcagatgaa gaacgcgtga aaggtctgat tgaagaaatg gcaagcgcct atgaagatcc
     1261 gaaagaagtg attgagttct atagcaaaaa caaagaactg atggacaaca tgcgtaatgt
     1321 tgcactggaa gaacaagccg ttgaagcagt tctggccaaa gcaaaagtga ccgaaaaaga
     1381 aaccaccttt aacgaactga tgaatcagca agccggtgca agcaccggat ccctggttcc
     1441 gcgtggtagc gctagcgaaa atctgtattt tcagggaatg gaaggtagca aaaccagcaa
     1501 taatagcacc atgcaggtta gctttgtttg tcagcgttgt agccagccgc tgaaactgga
     1561 taccagcttt aaaatcctgg atcgtgttac cattcaagaa ctgaccgcac cgctgctgac
     1621 caccgcacag gcaaaaccgg gtgaaaccca agaagaagaa accaattcag gcgaagaacc
     1681 gtttattgaa acaccgcgtc aggatggtgt tagccgtcgt tttattccgc ctgcacgtat
     1741 gatgagcacc gaaagcgcaa atagctttac cctgattggt gaagcaagtg atggtggcac
     1801 catggaaaat ctgagccgtc gtctgaaagt taccggtgac ctgtttgata tcatgagcgg
     1861 tcagaccgat gttgatcatc cgctgtgtga agaatgtacc gataccctgc tggatcagct
     1921 ggatacccaa ctgaatgtta ccgaaaatga atgccagaac tataaacgct gtctggaaat
     1981 tctggaacaa atgaacgaag atgatagcga acagctgcag atggaactga aagaactggc
     2041 actggaagaa gaacgtctga tccaagaact ggaagatgtg gaaaaaaacc gtaaaattgt
     2101 ggccgagaat ctggaaaaag ttcaggcaga agccgaacgc ctggatcaag aagaggcaca
     2161 gtatcagcgt gaatatagcg aatttaaacg tcagcagctg gaactggacg atgaactgaa
     2221 aagcgctgaa aatcaggcgc gttatgcaca gacccaggcg gacaaactga aaaaaacgaa
     2281 tgtttttaac gccacctttc acatttggca tagtggtcag tttggcacca ttaataactt
     2341 tcgtctgggt cgtctgccga gcgttccggt tgaatggaat gaaattaatg cagcatgggg
     2401 tcagacagtt ctgctgctgc atgcactggc caataaaatg ggtctgaaat tccagcgtta
     2461 tcgtctggtt ccgtatggta atcatagcta tctggaaagc ctgaccgata aaagcaaaga
     2521 actgccgctg tattgtagcg gtggtctgcg ttttttttgg gataacaaat ttgatcatgc
     2581 catggtggca tttctggatt gtgttcagca gtttaaagaa gaggttgaaa aaggcgaaac
     2641 ccgtttttgt ctgccgtatc gtatggatgt agaaaaaggc aaaattgaag ataccggtgg
     2701 tagtggtggt agctatagca ttaaaaccca gtttaatagc gaagaacagt ggaccaaagc
     2761 cctgaaattt atgctgacca atctgaaatg gggtctggca tgggttagca gccagtttta
     2821 taacaaataa agcctcgagg gtggtggtag cgattataaa gatgatgatg ataaaggcct
     2881 gaacgatatc tttgaagccc agaaaattga atggcacgag taaaagcttg tcgagcacca
     2941 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     3001 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     3061 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     3121 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3181 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3241 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3301 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3361 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3421 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3481 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3541 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3601 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3661 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3721 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3781 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3841 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3901 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3961 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4021 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4081 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4141 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     4201 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     4261 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     4321 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     4381 ataaatctgg agccggtgag cgtggtagtc gcggtatcat tgcagcactg gggccagatg
     4441 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4501 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4561 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4621 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4681 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4741 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4801 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4861 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4921 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4981 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5041 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5101 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5161 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5221 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5281 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5341 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5401 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5461 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     5521 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     5581 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     5641 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5701 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5761 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     5821 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5881 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5941 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     6001 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     6061 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     6121 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     6181 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     6241 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     6301 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     6361 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     6421 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     6481 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     6541 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     6601 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     6661 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     6721 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6781 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     6841 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     6901 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     6961 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     7021 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     7081 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     7141 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     7201 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     7261 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     7321 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     7381 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     7441 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     7501 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     7561 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     7621 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     7681 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     7741 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     7801 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     7861 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     7921 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     7981 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     8041 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     8101 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     8161 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaat
//
