LOCUS       sequence-350014-        7064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of PNR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7064)
  AUTHORS   Tan MH, Zhou XE, Soon FF, Li X, Li J, Yong EL, Melcher K, Xu HE
  TITLE     The crystal structure of the orphan nuclear receptor NR2E3/PNR 
            ligand binding domain reveals a dimeric auto-repressed conformation.
  JOURNAL   PLoS One. 2013 Sep 12;8(9):e74359. doi: 
            10.1371/journal.pone.0074359. eCollection 2013.
  PUBMED    24069298
REFERENCE   2  (bases 1 to 7064)
  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: "PLoS One.";
            date: "2013-09-12"; volume: "8(9):e74359. doi"; pages: " 
            10.1371/journal.pone.0074359. eCollection 2013"
COMMENT     Sequence Label: pETDuet1-MBP-NAAEF-PNR(217-410 C275)-His6
FEATURES             Location/Qualifiers
     source          1..7064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     206..228
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(222..413)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(985..2067)
                     /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     2029..2048
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2068..2145)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2351..2370
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2458..2476
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2477..2501
                     /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             2516..2538
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2545..3648
                     /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="MAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKL
                     EEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGK
                     LIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLI
                     AADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKG
                     ETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEF
                     LENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA
                     FWYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     3622..3645
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4249..4266
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        4335..4353
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4354..4378
                     /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)."
     CDS             4487..4531
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4569..4587)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4583..4630
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4667..5122
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4754..4773)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4964..4985
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5237..6028,6029..6097))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5860..5879
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6098..6190)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6271..6859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6760..6779
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7013..7030
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(7045..7064,1..120)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
      121 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
      181 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
      241 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
      301 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
      361 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
      421 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
      481 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
      541 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
      601 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
      661 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
      721 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
      781 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
      841 cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca ccggaaggag
      901 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
      961 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1021 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     1081 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     1141 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     1201 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     1261 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     1321 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     1381 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     1441 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     1501 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     1561 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     1621 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     1681 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     1741 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     1801 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     1861 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     1921 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     1981 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     2041 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     2101 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     2161 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     2221 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     2281 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     2341 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     2401 tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa
     2461 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     2521 ttaactttaa gaaggagata taccatggca aaaatcgaag aaggtaaact ggtaatctgg
     2581 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     2641 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     2701 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     2761 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     2821 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     2881 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     2941 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     3001 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     3061 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     3121 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     3181 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     3241 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     3301 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     3361 ccgaacaaag agctggcgaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     3421 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     3481 ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     3541 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     3601 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa cgcggcggcc
     3661 gaattcctgg acagcatcca tgagacctcg gctcgcctac tcttcatggc cgtcaagtgg
     3721 gccaagaacc tgcctgtgtt ctccagcctg cccttccggg atcaggtgat cctgctggaa
     3781 gaggcgtgga gtgaactctt tctcctcggg gccatccagt ggtctctgcc tctggacagc
     3841 agtcctctgc tggcaccgcc cgaggcctct gctgccggtg gtgcccaggg ccggctcacg
     3901 ctggccagca tggagacgcg tgtcctgcag gaaactatct ctcggttccg ggcattggcg
     3961 gtggacccca cggagtttgc ctgcatgaag gccttggtcc tcttcaagcc agagacgcgg
     4021 ggcctgaagg atcctgagca cgtagaggcc ttgcaggacc agtcccaagt gatgctgagc
     4081 cagcacagca aggcccacca ccccagccag cccgtgaggt ttgggaaatt gctcctgctc
     4141 ctcccgtctt tgaggtttat cactgcggaa cgcatcgagc tcctcttttt ccgcaagacc
     4201 atagggaata ctccaatgga gaagctcctt tctgatatgt tcaaaaacca tcaccatcac
     4261 catcactgag cggccgcata atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc
     4321 cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     4381 atcttagtat attagttaag tataagaagg agatatacat atggcagatc tcaattggat
     4441 atcggccggc cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag aaaccgctgc
     4501 tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc
     4561 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     4621 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     4681 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4741 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4801 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4861 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     4921 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4981 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5041 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5101 aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct
     5161 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5221 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5281 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5341 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5401 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5461 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5521 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5581 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5641 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5701 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5761 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5821 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5881 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5941 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6001 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6061 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat
     6121 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6181 aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6241 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6301 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6361 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     6421 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6481 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6541 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6601 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6661 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6721 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6781 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6841 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6901 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6961 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     7021 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctcc
//
