LOCUS       sequence-193154-        7330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of NRBF2, which associates with the PI3KC3-C1 
            complex.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7330)
  AUTHORS   Young LN, Cho K, Lawrence R, Zoncu R, Hurley JH
  TITLE     Dynamics and architecture of the NRBF2-containing 
            phosphatidylinositol 3-kinase complex I of autophagy.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Jul 19;113(29):8224-9. doi: 
            10.1073/pnas.1603650113. Epub 2016 Jul 6.
  PUBMED    27385829
REFERENCE   2  (bases 1 to 7330)
  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: 
            "10.1073/pnas.1603650113"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2016-07-19- 19"; volume: "113"; issue: "29"; pages: "8224-9"
COMMENT     Sequence Label: His-MBP-NRBF2
FEATURES             Location/Qualifiers
     source          1..7330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     63..82
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        63..81
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    82..106
                     /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             121..143
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             162..179
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             189..1289
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1263..1286
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1302..1322
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2256..2273
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2326..2344)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2340..2387
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2424..2879
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2511..2530)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2721..2742
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2972..3787)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2972..3787)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3695..3714
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3779..3798
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3909..4497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4398..4417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4651..4668
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4683..4822
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4748..4767)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4908..4930
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4924..5115)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5924..7006)
                     /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     6968..6987
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7007..7084)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7290..7309
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
       61 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
      121 tttgtttaac tttaagaagg agatatacca tgggttcttc tcaccatcac catcaccatg
      181 gttcttctat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
      241 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
      301 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
      361 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
      421 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
      481 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
      541 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
      601 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
      661 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
      721 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
      781 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
      841 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
      901 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
      961 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     1021 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     1081 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     1141 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     1201 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     1261 tcgatgaagc cctgaaagac gcgcagacta atgggatcga ggaaaacctg tacttccaat
     1321 ccaatgcaga agtaatggaa ggacccctca acctggctca tcaacagagc agacgagcag
     1381 accgtttatt agctgcaggc aaatacgaag aggctatttc ttgtcacaaa aaggctgcag
     1441 catatctttc tgaagccatg aagctgacac agtcagagca ggctcatctt tcactggaat
     1501 tgcaaaggga tagccatatg aaacagctcc tcctcatcca agagagatgg aaaagggccc
     1561 agcgtgaaga aagattgaaa gcccagcaga acacagacaa ggatgcagct gcccatcttc
     1621 agacatctca caaaccctct gcagaggatg cagagggcca gagtcccctt tctcagaagt
     1681 acagcccttc cacagagaaa tgcctgcctg agattcaggg gatctttgac agggatccag
     1741 acacactact ttatttactt cagcaaaaga gtgagccagc agagccatgt attggaagca
     1801 aagccccaaa agatgataaa acaattatag aggagcaggc aaccaaaatt gcagatttga
     1861 agaggcatgt ggaattcctt gtggctgaga atgaaagatt aaggaaagaa aataaacaac
     1921 taaaggctga aaaggccaga cttctaaaag gtccaataga aaaggagctg gatgtagatg
     1981 ctgattttgt agaaacgtca gagttatgga gcttgccacc acatgcagaa actgctacag
     2041 cctcctcaac ctggcagaag ttcgcagcaa atactgggaa agccaaggac attccaatcc
     2101 ccaatcttcc tcccttggat tttccatctc cagaacttcc tcttatggag ctctctgagg
     2161 atattctgaa aggatttatg aataattaat aacattggaa gtggataacg gatccgaatt
     2221 cgagcgccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
     2281 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     2341 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2401 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2461 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2521 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2581 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2641 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2701 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2761 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2821 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa ctagtaacgt ttacaatttc
     2881 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2941 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     3001 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3061 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3121 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3181 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     3241 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3301 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3361 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3421 catcaacaat gttttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3481 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3541 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3601 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3661 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3721 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3781 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4021 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4561 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4621 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4681 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4741 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4801 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4861 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4921 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4981 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5041 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     5101 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5161 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     5221 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5281 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5341 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5401 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5461 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5521 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5581 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5641 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5701 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5761 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5821 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5881 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5941 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6001 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     6061 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     6121 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     6181 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     6241 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatac
     6301 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     6361 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     6421 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     6481 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     6541 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     6601 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     6661 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6721 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     6781 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     6841 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     6901 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     6961 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     7021 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     7081 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     7141 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     7201 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     7261 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     7321 gcacctgtgg
//
