LOCUS       sequence-266255-        6324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PPI (BiFC/TriFC) assay vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6324)
  TITLE     Lewin Lab Plasmids
REFERENCE   2  (bases 1 to 6324)
  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"
COMMENT     Sequence Label: pRDAX-149    N-Tag GFP[2] L32 FRB EBPF2
FEATURES             Location/Qualifiers
     source          1..6324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        151..530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        531..734
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     685..705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     731..755
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          870..1002
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1017..1026
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1191..1466
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS"
     misc_feature    join(1483..2057,2058..2060,2061..2069)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1650..1667)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1877..1896
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2070..2072,2073..2075,2076..2789)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(2115..2136)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     intron          2835..2967
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_feature    2976..3564
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3029..3049)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3447..3458)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3591..3608)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3597..3708
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3804..3824)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3804..3822)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3843..3859)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3843..3859)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3856..3878)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(3961..3980)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4026..4081
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4080..4099)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     rep_origin      complement(4460..5048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4540..4559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5219..6010,6011..6079))
                     /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     5842..5861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6080..6184)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6252..6270
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctgcctccag gcctcaatat tggccattag ccatattatt cattggttat atagcataaa
       61 tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg tacatttata
      121 ttggctcatg tccaatatga ccgccatgtt ggcattgatt attgactagt tattaatagt
      181 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      241 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      301 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      361 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta
      421 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
      481 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      541 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      601 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      661 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      721 atataagcag agctcgttta gtgaaccgtc agatcactag aagctttatt gcggtagttt
      781 atcacagtta aattgctaac gcagtcagtg cttctgacac aacagtctcg aacttaagct
      841 gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag
      901 gagaccaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac
      961 ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac tcccaggcca
     1021 ccatggaaaa gagggaccac atggtgctgc tcgagtacgt cacagcagct ggcatcaccg
     1081 atgccagcga cgtgggtggc ggagggagcg aaggcggcgg atccggaggg cctggctctg
     1141 gcggagaggg gagcgctgga ggcgggtcag ccggtggtgg gagtgtggcc atcctctggc
     1201 atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg
     1261 tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga
     1321 ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt
     1381 ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cacccaagcc tgggacctct
     1441 attatcatgt gttccgacga atctcataag aattcagatc cgcccctctc cctccccccc
     1501 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     1561 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     1621 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     1681 tgtcgtgaag gaagcagttc ctctggtagc ttcttgaaga caaacaacgt ctgtagcgac
     1741 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     1801 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     1861 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     1921 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tacacatgct ttacatgtgt
     1981 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     2041 aaaaacacga tgataatatg gccacaacca tggtgagcaa gggcgaggag ctgttcaccg
     2101 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtga
     2161 ggggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca
     2221 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgagccac ggcgtgcagt
     2281 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2341 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcacctac aagacccgcg
     2401 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcgtcgact
     2461 tcaaggagga cggcaacatc ctggggcaca agctggagta caacttcaac agccacaaca
     2521 tctatatcat ggccgtcaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2581 acgtggagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2641 acggccccgt gctgctgccc gacagccact acctgagcac ccagtccgtg ctgagcaaag
     2701 accccaacga gaagcgcgat cacatggtcc tgctggagtt ccgcaccgcc gccgggatca
     2761 ctctcggcat ggacgagctg tacaagtaaa ccggtgctcg aggtaccgat gaatagctaa
     2821 ggtcgaggcc gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac
     2881 tgggcttgtc gagacagaga agactcttgc gtttctgata ggcacctatt ggtcttactg
     2941 acatccactt tgcctttctc tccacaggtg tcgacaatca acctctggat tacaaaattt
     3001 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     3061 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     3121 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     3181 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     3241 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     3301 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     3361 tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc
     3421 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     3481 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     3541 tctccctttg ggccgcctcc ccgcctggaa ttcgagctcg gtacgatcag cctcgactgt
     3601 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3661 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3721 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3781 agacaatagc ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     3841 tggtcatagc tgtttcctgt gtgaaattgt tatccgctaa ttcactcctc aggtgcaggc
     3901 tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga
     3961 tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc
     4021 tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc
     4081 actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta
     4141 gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg
     4201 tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac
     4261 ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa
     4321 attttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat
     4381 agctgtccct cttctcttat ggagatcggc cggccgccgg cgtcgacgcg cgcggccgcg
     4441 gcgcgccgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4501 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4561 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4621 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4681 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4741 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4801 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4861 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4921 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4981 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5041 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5101 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5161 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5221 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5281 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5341 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5401 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5461 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5521 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5581 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5641 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5701 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5761 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5821 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5881 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5941 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6001 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6061 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6121 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6181 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6241 taacctataa aaataggcgt atcacgaggc cctttcgtct acgcgcgcgc cggcggcgcg
     6301 ccgtcgacgg ccggccgcgg ccgc
//
