LOCUS       sequence-204859-        7690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7690)
  AUTHORS   Chinthalapudi K, Mandati V, Zheng J, Sharff AJ, Bricogne G, Griffin 
            PR, Kissil J, Izard T
  TITLE     Lipid binding promotes the open conformation and tumor-suppressive 
            activity of neurofibromin 2.
  JOURNAL   Nat Commun. 2018 Apr 6;9(1):1338. doi: 10.1038/s41467-018-03648-4.
  PUBMED    29626191
REFERENCE   2  (bases 1 to 7690)
  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: "Nat 
            Commun."; date: "2018-04-6"; volume: "9(1):1338. doi"; pages: " 
            10.1038/s41467-018-03648-4"
COMMENT     Sequence Label: pET32a-Trx-PP-hNF2
FEATURES             Location/Qualifiers
     source          1..7690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      34..622
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     523..542
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     776..793
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    808..947
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(873..892)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1033..1055
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1049..1240)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2049..3131)
                     /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     3093..3112
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3132..3209)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3415..3434
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3522..3541
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3522..3540
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3541..3565
                     /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             3580..3602
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3610..3936
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             3958..3975
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3985..4008
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5803..5847
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             5863..5877
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             5939..5956
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6009..6027)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6023..6070
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6107..6562
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6194..6213)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6404..6425
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6588..6692
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6693..6761,6762..7553)
                     /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(6911..6930)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
       61 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
      121 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
      181 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
      241 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      301 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      361 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
      421 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      481 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      541 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      601 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      661 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      721 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      781 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
      841 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
      901 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
      961 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     1021 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     1081 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     1141 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     1201 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     1261 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     1321 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     1381 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     1441 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     1501 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     1561 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     1621 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     1681 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     1741 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     1801 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     1861 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     1921 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     1981 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     2041 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2101 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     2161 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     2221 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     2281 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     2341 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     2401 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     2461 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     2521 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     2581 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     2641 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     2701 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     2761 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     2821 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     2881 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     2941 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     3001 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     3061 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     3121 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     3181 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     3241 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     3301 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     3361 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     3421 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     3481 tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg
     3541 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     3601 gatatacata tgagcgataa aattattcac ctgactgacg acagttttga cacggatgta
     3661 ctcaaagcgg acggggcgat cctcgtcgat ttctgggcag agtggtgcgg tccgtgcaaa
     3721 atgatcgccc cgattctgga tgaaatcgct gacgaatatc agggcaaact gaccgttgca
     3781 aaactgaaca tcgatcaaaa ccctggcact gcgccgaaat atggcatccg tggtatcccg
     3841 actctgctgc tgttcaaaaa cggtgaagtg gcggcaacca aagtgggtgc actgtctaaa
     3901 ggtcagttga aagagttcct cgacgctaac ctggccggtt ctggttctgg ccatatgcac
     3961 catcatcatc atcattcttc tggtctggaa gttctgttcc aggggcccat ggccggggcc
     4021 atcgcttccc gcatgagctt cagctctctc aagaggaagc aacccaagac gttcaccgtg
     4081 aggatcgtca ccatggacgc cgagatggag ttcaattgcg agatgaagtg gaaagggaag
     4141 gacctctttg atttggtgtg ccggactctg gggctccgag aaacctggtt ctttggactg
     4201 cagtacacaa tcaaggacac agtggcctgg ctcaaaatgg acaagaaggt actggatcat
     4261 gatgtttcaa aggaagaacc agtcaccttt cacttcttgg ccaaatttta tcctgagaat
     4321 gctgaagagg agctggttca ggagatcaca caacatttat tcttcttaca ggtaaagaag
     4381 cagattttag atgaaaagat ctactgccct cctgaggctt ctgtgctcct ggcttcttac
     4441 gccgtccagg ccaagtatgg tgactacgac cccagtgttc acaagcgggg atttttggcc
     4501 caagaggaat tgcttccaaa aagggtaata aatctgtatc agatgactcc ggaaatgtgg
     4561 gaggagagaa ttactgtttg gtacgcagag caccgaggcc gagccaggga tgaagctgaa
     4621 atggaatatc tgaagatagc tcaggacctg gagatgtacg gtgtgaacta ctttgcaatc
     4681 cggaataaaa agggcacaga gctgctgctt ggagtggatg ccctggggct tcacatttat
     4741 gaccctgaga acagactgac ccccaagatc tccttcccgt ggaatgaaat ccgaaacatc
     4801 tcgtacagtg acaaggagtt tactattaaa ccactggata agaaaattga tgtcttcaag
     4861 tttaactcct caaagcttcg tgttaataag ctgattctcc agctatgtat cgagaaccat
     4921 gatctattta tgaggagaag gaaagccgat tctttggaag ttcagcagat gaaagcccag
     4981 gccagggagg agaaggctag aaagcagatg gagcggcagc gcctcgctcg agagaagcag
     5041 atgagggagg aggctgaacg cacgagggat gagttggaga ggaggctgct gcagatgaaa
     5101 gaagaagcaa caatggccaa cgaagcactg atgcggtctg aggagacagc tgacctgttg
     5161 gctgaaaagg cccagatcac cgaggaggag gcaaaacttc tggcccagaa ggccgcagag
     5221 gctgagcagg aaatgcagcg catcaaggcc acagcgattc gcacggagga ggagaagcgc
     5281 ctgatggagc agaaggtgct ggaagccgag gtgctggcac tgaagatggc tgaggagtca
     5341 gagaggaggg ccaaagaggc agatcagctg aagcaggacc tgcaggaagc acgcgaggcg
     5401 gagcgaagag ccaagcagaa gctcctggag attgccacca agcccacgta cccgcccatg
     5461 aacccaattc cagcaccgtt gcctcctgac ataccaagct tcaacctcat tggtgacagc
     5521 ctgtctttcg acttcaaaga tactgacatg aagcggcttt ccatggagat agagaaagaa
     5581 aaagtggaat acatggaaaa gagcaagcat ctgcaggagc agctcaatga actcaagaca
     5641 gaaatcgagg ccttgaaact gaaagagagg gagacagctc tggatattct gcacaatgag
     5701 aactccgaca gaggtggcag cagcaagcac aataccatta aaaagctcac cttgcagagc
     5761 gccaagtccc gagtggcctt ctttgaagag ctctagggta tgaaagaaac cgctgctgct
     5821 aaattcgaac gccagcacat ggacagccca gatctgggta ccgacgacga cgacaaggcc
     5881 atggctgata tcggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca
     5941 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     6001 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     6061 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     6121 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     6181 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     6241 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     6301 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     6361 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     6421 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     6481 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     6541 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     6601 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     6661 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6721 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6781 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6841 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     6901 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     6961 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     7021 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     7081 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     7141 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     7201 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     7261 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     7321 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     7381 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     7441 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     7501 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     7561 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     7621 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     7681 tccactgagc
//
