LOCUS       sequence-249275-        6809 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 6809)
  TITLE     Lewin Lab Plasmids
REFERENCE   2  (bases 1 to 6809)
  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-212    N-Tag miRFP670[2] L20 FRB EGFP
FEATURES             Location/Qualifiers
     source          1..6809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        153..532
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        533..736
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     687..707
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     733..757
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          872..1004
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1019..1028
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1676..1951
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS"
     misc_feature    join(1968..2542,2543..2545,2546..2554)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2135..2152)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2362..2381
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2555..2557,2558..2560,2561..3274)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2555..2557,2558..2560,2561..3271)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2600..2621)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2861..2880)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3208..3229
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          3320..3452
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_feature    3461..4049
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3514..3534)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3932..3943)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4076..4093)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4082..4193
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4289..4309)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4289..4307)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4328..4344)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4328..4344)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4341..4363)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(4446..4465)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4511..4566
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4565..4584)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     rep_origin      complement(4945..5533)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5025..5044)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5704..6495,6496..6564))
                     /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     6327..6346
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6565..6669)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6737..6755
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcctgcctcc aggcctcaat attggccatt agccatatta ttcattggtt atatagcata
       61 aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta
      121 tattggctca tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaata
      181 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      241 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      301 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      361 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc
      421 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg
      481 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      541 gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
      601 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
      661 atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt
      721 ctatataagc agagctcgtt tagtgaaccg tcagatcact agaagcttta ttgcggtagt
      781 ttatcacagt taaattgcta acgcagtcag tgcttctgac acaacagtct cgaacttaag
      841 ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta
      901 aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc
      961 acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccaggc
     1021 caccatgggc agagcgggac ccagtatcga cctgtctggg acactcgcac cagcactgga
     1081 gcgcataaga actgctggat cactgagggc cctctgtgac gacacagtct tgctctttca
     1141 gcaatgtacg ggctatgatc gagtgatggt gtaccgattc gacgaacagg gtcatggtct
     1201 ggtcttttcc gagtgtcacg tgcctggcct ggagagctac tttggcaacc gctatccctc
     1261 ttctaccgta ccccagatgg ccaggcaact ttacgtccgg caacgggtca gagtgctggt
     1321 ggatgtgacc taccagcctg tgccactcga acctcggctg agcccactca ctggacggga
     1381 tctggacatg agcggctgct tcctgaggtc catgtcacct tgccatctgc agttcttgaa
     1441 ggacatgggc gttcgagcca cactcgcagt ctcactggtt gtaggcggga aactgtgggg
     1501 tcttgtggtg tgtcaccact accttcccag gttcatccgg ttcgagttga gagccatctg
     1561 caaacgtttg gccgagagaa tagccacccg cattactgct ctggaatccg ggagtcctgg
     1621 cggtggatcc ggaggctctg ggagctctgc cagcggtggc agcacctccg tggccatcct
     1681 ctggcatgag atgtggcatg aaggcctgga agaggcatct cgtttgtact ttggggaaag
     1741 gaacgtgaaa ggcatgtttg aggtgctgga gcccttgcat gctatgatgg aacggggccc
     1801 ccagactctg aaggaaacat cctttaatca ggcctatggt cgagatttaa tggaggccca
     1861 agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag gacctcaccc aagcctggga
     1921 cctctattat catgtgttcc gacgaatctc ataagaattc agatccgccc ctctccctcc
     1981 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     2041 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     2101 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     2161 ttgaatgtcg tgaaggaagc agttcctctg gtagcttctt gaagacaaac aacgtctgta
     2221 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     2281 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     2341 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     2401 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtacac atgctttaca
     2461 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     2521 ctttgaaaaa cacgatgata atatggccac aaccatggtg agcaagggcg aggagctgtt
     2581 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2641 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     2701 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     2761 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     2821 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     2881 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     2941 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     3001 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     3061 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     3121 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     3181 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     3241 gatcactctc ggcatggacg agctgtacaa gtaaaccggt gctcgaggta ccgatgaata
     3301 gctaaggtcg aggccgcagg taagtatcaa ggttacaaga caggtttaag gagaccaata
     3361 gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac ctattggtct
     3421 tactgacatc cactttgcct ttctctccac aggtgtcgac aatcaacctc tggattacaa
     3481 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3541 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3601 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     3661 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3721 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     3781 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     3841 ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat
     3901 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     3961 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     4021 tcggatctcc ctttgggccg cctccccgcc tggaattcga gctcggtacg atcagcctcg
     4081 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     4141 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     4201 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     4261 tgggaagaca atagcccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     4321 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctaattcac tcctcaggtg
     4381 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     4441 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     4501 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     4561 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     4621 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     4681 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     4741 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     4801 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     4861 gtcatagctg tccctcttct cttatggaga tcggccggcc gccggcgtcg acgcgcgcgg
     4921 ccgcggcgcg ccgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4981 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5041 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5101 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5161 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5221 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5281 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5341 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5401 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5461 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5521 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5581 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5641 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5701 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5761 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5821 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5881 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5941 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6001 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6061 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6121 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6181 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6241 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6301 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6361 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6421 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6481 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6541 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6601 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6661 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     6721 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctacgcg cgcgccggcg
     6781 gcgcgccgtc gacggccggc cgcggccgc
//
