LOCUS       sequence-191826-        6946 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the monomeric form of NRBF2 that binds with 
            the PI3K C1 complex.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6946)
  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 6946)
  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 (1-159)
FEATURES             Location/Qualifiers
     source          1..6946
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..1227
                     /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     1201..1224
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1240..1260
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1810..1827
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1880..1898)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1894..1941
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1978..2433
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2065..2084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2275..2296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2526..3341)
                     /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(2526..3341)
                     /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     3249..3268
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3333..3352
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3463..4051
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3952..3971
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4205..4222
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4237..4376
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4302..4321)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4462..4484
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4478..4669)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5478..6560)
                     /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     6522..6541
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6561..6638)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6844..6863
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggttcttctc accatcacca tcaccatggt
      121 tcttctatga aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat
      181 aacggtctcg ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt
      241 gagcatccgg ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct
      301 gacattatct tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct
      361 gaaatcaccc cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta
      421 cgttacaacg gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat
      481 aacaaagatc tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa
      541 gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc
      601 tggccgctga ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac
      661 attaaagacg tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac
      721 ctgattaaaa acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt
      781 aataaaggcg aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc
      841 agcaaagtga attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg
      901 ttcgttggcg tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa
      961 gagttcctcg aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa
     1021 ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt
     1081 gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc
     1141 gctttctggt atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc
     1201 gatgaagccc tgaaagacgc gcagactaat gggatcgagg aaaacctgta cttccaatcc
     1261 aatgcagaag taatggaagg acccctcaac ctggctcatc aacagagcag acgagcagac
     1321 cgtttattag ctgcaggcaa atacgaagag gctatttctt gtcacaaaaa ggctgcagca
     1381 tatctttctg aagccatgaa gctgacacag tcagagcagg ctcatctttc actggaattg
     1441 caaagggata gccatatgaa acagctcctc ctcatccaag agagatggaa aagggcccag
     1501 cgtgaagaaa gattgaaagc ccagcagaac acagacaagg atgcagctgc ccatcttcag
     1561 acatctcaca aaccctctgc agaggatgca gagggccaga gtcccctttc tcagaagtac
     1621 agcccttcca cagagaaatg cctgcctgag attcagggga tctttgacag ggatccagac
     1681 acactacttt atttacttca gcaaaagagt gagccagcag agccatgtat tggaagcaaa
     1741 taataacatt ggaagtggat aacggatccg aattcgagcg ccgtcgacaa gcttgcggcc
     1801 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     1861 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1921 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     1981 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2041 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2101 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2161 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2221 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2281 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2341 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2401 cgaattttaa caaactagta acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc
     2461 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     2521 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2581 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2641 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2701 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2761 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2821 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2881 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2941 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatgttttc acctgaatca
     3001 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     3061 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     3121 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     3181 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     3241 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3301 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     3361 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     3421 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3481 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3541 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3601 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3661 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3721 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3781 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3841 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3901 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3961 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4021 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4081 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4141 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4201 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     4261 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     4321 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     4381 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4441 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4501 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     4561 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4621 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4681 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4741 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4801 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4861 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4921 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4981 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     5041 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     5101 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     5161 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     5221 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     5281 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     5341 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     5401 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     5461 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5521 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5581 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5641 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     5701 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     5761 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     5821 tcgttggcaa ccagcatcgc agtgggaacg ataccctcat tcagcatttg catggtttgt
     5881 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5941 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     6001 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     6061 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     6121 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     6181 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     6241 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     6301 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     6361 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     6421 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     6481 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6541 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6601 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     6661 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     6721 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     6781 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     6841 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     6901 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaat
//
