LOCUS       sequence-249277-        6845 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 6845)
  TITLE     Lewin Lab Plasmids
REFERENCE   2  (bases 1 to 6845)
  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-214    N-Tag miRFP670[2] L32 FRB EGFP
FEATURES             Location/Qualifiers
     source          1..6845
                     /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             1712..1987
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS"
     misc_feature    join(2004..2578,2579..2581,2582..2590)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2171..2188)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2398..2417
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2591..2593,2594..2596,2597..3310)
                     /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(2591..2593,2594..2596,2597..3307)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2636..2657)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2897..2916)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3244..3265
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          3356..3488
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_feature    3497..4085
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3550..3570)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3968..3979)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4112..4129)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4118..4229
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4325..4345)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4325..4343)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4364..4380)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4364..4380)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4377..4399)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(4482..4501)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4547..4602
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4601..4620)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     rep_origin      complement(4981..5569)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5061..5080)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5740..6531,6532..6600))
                     /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     6363..6382
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6601..6705)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6773..6791
                     /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 acgtgggtgg
     1621 cggagggagc gaaggcggcg gatccggagg gcctggctct ggcggagagg ggagcgctgg
     1681 aggcgggtca gccggtggtg ggagtgtggc catcctctgg catgagatgt ggcatgaagg
     1741 cctggaagag gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt
     1801 gctggagccc ttgcatgcta tgatggaacg gggcccccag actctgaagg aaacatcctt
     1861 taatcaggcc tatggtcgag atttaatgga ggcccaagag tggtgcagga agtacatgaa
     1921 atcagggaat gtcaaggacc tcacccaagc ctgggacctc tattatcatg tgttccgacg
     1981 aatctcataa gaattcagat ccgcccctct ccctcccccc cccctaacgt tactggccga
     2041 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     2101 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     2161 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     2221 cctctggtag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     2281 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     2341 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     2401 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     2461 ggatctgatc tggggcctcg gtacacatgc tttacatgtg tttagtcgag gttaaaaaaa
     2521 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat
     2581 ggccacaacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2641 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     2701 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2761 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2821 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2881 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     2941 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3001 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     3061 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3121 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3181 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     3241 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3301 gtacaagtaa accggtgctc gaggtaccga tgaatagcta aggtcgaggc cgcaggtaag
     3361 tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag
     3421 aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct
     3481 ctccacaggt gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     3541 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     3601 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     3661 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     3721 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     3781 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     3841 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc
     3901 ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     3961 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     4021 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     4081 cccgcctgga attcgagctc ggtacgatca gcctcgactg tgccttctag ttgccagcca
     4141 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4201 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4261 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag cccagctttt
     4321 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     4381 tgtgaaattg ttatccgcta attcactcct caggtgcagg ctgcctatca gaaggtggtg
     4441 gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa
     4501 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
     4561 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
     4621 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
     4681 catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag
     4741 ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt
     4801 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
     4861 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
     4921 tggagatcgg ccggccgccg gcgtcgacgc gcgcggccgc ggcgcgccgt tgctggcgtt
     4981 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5041 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5101 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5161 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5221 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5281 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5341 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5401 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5461 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5521 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5581 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5641 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5701 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5761 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5821 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5881 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5941 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6001 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6061 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6121 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6181 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6241 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6301 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6361 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6421 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6481 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6541 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6601 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6661 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6721 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     6781 tatcacgagg ccctttcgtc tacgcgcgcg ccggcggcgc gccgtcgacg gccggccgcg
     6841 gccgc
//
