LOCUS       sequence-223022-        7378 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRB-TEV-N.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7378)
  AUTHORS   Caldwell RM, Bermudez JG, Thai D, Aonbangkhen C, Schuster BS, 
            Courtney T, Deiters A, Hammer DA, Chenoweth DM, Good MC
  TITLE     Optochemical Control of Protein Localization and Activity within 
            Cell-like Compartments.
  JOURNAL   Biochemistry. 2018 May 8;57(18):2590-2596. doi: 
            10.1021/acs.biochem.8b00131. Epub 2018 Apr 19.
  PUBMED    29671583
REFERENCE   2  (bases 1 to 7378)
  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.8b00131"; journalName: "Biochemistry"; date: 
            "2018-05-8- 8"; volume: "57"; issue: "18"; pages: "2590-2596"
COMMENT     Sequence Label: pRC04
FEATURES             Location/Qualifiers
     source          1..7378
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(841..860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1001..1023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1017..1208)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        1704..1781
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             1782..2864
                     /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     complement(1801..1820)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3107..3112,3113..3128,3129..3135)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3143..3159
                     /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)."
     primer_bind     3148..3170
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             3229..4329
                     /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="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      4186..4195
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     4303..4326
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4378..4389
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4432..4452
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4459..4728
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             5128..5145
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5172..5189)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5172..5188)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5181..5203)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5367..5384)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5537..5623
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5715..5742
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5761..5852
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5853..5921,5922..6713)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6071..6090)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6755..7268)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6950..6971)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7162..7181
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
      841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1021 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     1081 tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     1141 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     1201 ttggtcactg atgcctccgt gtaatgggga tttctgttca tgggggtaat gataccgatg
     1261 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     1321 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     1381 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     1441 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     1501 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     1561 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     1621 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag
     1681 gacccaacgc tgcccgaaat tccgacacca tcgaatggtg caaaaccttt cgcggtatgg
     1741 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     1801 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     1861 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     1921 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     1981 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     2041 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     2101 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     2161 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     2221 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     2281 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     2341 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     2401 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     2461 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     2521 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     2581 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     2641 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     2701 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     2761 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     2821 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     2881 gtaagttagc tcactcatta ggcacaattc tcatgtttga cagcttatca tcgactgcac
     2941 ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt
     3001 cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt
     3061 tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca
     3121 tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagccag
     3181 tccgtttagg tgttttcacg agcacttcac caacaaggac catagattat gaaaactgaa
     3241 gaaggtaaac tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc
     3301 ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg
     3361 gaagagaaat tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca
     3421 cacgaccgct ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa
     3481 gcgttccagg acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg
     3541 attgcttacc cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg
     3601 aacccgccaa aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt
     3661 aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct
     3721 gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg
     3781 gataacgctg gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac
     3841 atgaatgcag acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg
     3901 atgaccatca acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt
     3961 gtaacggtac tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc
     4021 gcaggtatta acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat
     4081 ctgctgactg atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg
     4141 ctgaagtctt acgaggaaga gttggcgaaa gatccacgta ttgccgccac catggaaaac
     4201 gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg
     4261 cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac
     4321 gcgcagacta attcgagctc gaacaacaac aacaataaca ataacaacaa cctcgggatc
     4381 gagggaagga tttcaactag tggatctggt ggtggtggcg gttccatgag cgagaatctt
     4441 tattttcagg gatccatgga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac
     4501 tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg
     4561 gaacggggcc cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta
     4621 atggaggccc aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcacc
     4681 caagcctggg acctctatta tcatgtgttc cgacgaatct caaagcaggg tggcggcggg
     4741 agtggaggag gtgggagtgg agaaagcttg tttaaggggc cgcgtgatta caacccgata
     4801 tcgagcacca tttgtcattt gacgaatgaa tctgatgggc acacaacatc gttgtatggt
     4861 attggatttg gtcccttcat cattacaaac aagcacttgt ttagaagaaa taatggaaca
     4921 ctgttggtcc aatcactaca tggtgtattc aaggtcaaga acaccacgac tttgcaacaa
     4981 cacctcattg atgggaggga catgataatt attcgcatgc ctaaggattt cccaccattt
     5041 cctcaaaagc tgaaatttag agagccacaa agggaagagc gcatatgtct tgtgacaacc
     5101 aacttccaaa cttgataaag cggccgccac catcaccacc accactaggg gccgcactcg
     5161 agaagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     5221 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     5281 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggca gcttggctgt
     5341 tttggcggat gagataagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     5401 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     5461 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     5521 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     5581 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     5641 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     5701 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct
     5761 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5821 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5881 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5941 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6001 acagcggtaa gatccttgag agttttcgcc ccgaagaacg tttcccaatg atgagcactt
     6061 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     6121 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     6181 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6241 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6301 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6361 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6421 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6481 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6541 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6601 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6661 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6721 accaagttta ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc
     6781 caaaaacagg aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa
     6841 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     6901 aatcccttat aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa
     6961 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     7021 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg
     7081 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     7141 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     7201 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     7261 gggcgcgtaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7321 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
