LOCUS       sequence-249364-        6290 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 6290)
  TITLE     Lewin Lab Plasmids
REFERENCE   2  (bases 1 to 6290)
  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-146    C-Tag GFP[2] L20 FRB EBPF2
FEATURES             Location/Qualifiers
     source          1..6290
                     /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"
     regulatory      1023..1032
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1034..1309
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS"
     misc_feature    join(1449..2023,2024..2026,2027..2035)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1616..1633)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1843..1862
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2036..2038,2039..2041,2042..2755)
                     /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(2081..2102)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     intron          2801..2933
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_feature    2942..3530
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2995..3015)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3413..3424)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3557..3574)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3563..3674
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3770..3790)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3770..3788)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3809..3825)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3809..3825)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3822..3844)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(3927..3946)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3992..4047
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4046..4065)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     rep_origin      complement(4426..5014)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4506..4525)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5185..5976,5977..6045))
                     /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     5808..5827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6046..6150)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6218..6236
                     /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 caccatggtg gccatcctct ggcatgagat gtggcatgaa ggcctggaag aggcatctcg
     1081 tttgtacttt ggggaaagga acgtgaaagg catgtttgag gtgctggagc ccttgcatgc
     1141 tatgatggaa cggggccccc agactctgaa ggaaacatcc tttaatcagg cctatggtcg
     1201 agatttaatg gaggcccaag agtggtgcag gaagtacatg aaatcaggga atgtcaagga
     1261 cctcacccaa gcctgggacc tctattatca tgtgttccga cgaatctcag ggagtcctgg
     1321 cggtggatcc ggaggctctg ggagctctgc cagcggtggc agcacctccg aaaagaggga
     1381 ccacatggtg ctgctcgagt acgtcacagc agctggcatc accgatgcca gctaagaatt
     1441 cagatccgcc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa
     1501 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg
     1561 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc
     1621 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggtagcttct
     1681 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac
     1741 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc
     1801 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta
     1861 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg
     1921 cctcggtaca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
     1981 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca caaccatggt
     2041 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2101 cgtaaacggc cacaagttca gcgtgagggg cgagggcgag ggcgatgcca ccaacggcaa
     2161 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2221 gaccaccctg agccacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     2281 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2341 ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2401 ccgcatcgag ctgaagggcg tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2461 ggagtacaac ttcaacagcc acaacatcta tatcatggcc gtcaagcaga agaacggcat
     2521 caaggtgaac ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca
     2581 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct
     2641 gagcacccag tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2701 ggagttccgc accgccgccg ggatcactct cggcatggac gagctgtaca agtaaaccgg
     2761 tgctcgaggt accgatgaat agctaaggtc gaggccgcag gtaagtatca aggttacaag
     2821 acaggtttaa ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt
     2881 ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcga
     2941 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     3001 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     3061 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     3121 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     3181 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     3241 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     3301 gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct
     3361 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3421 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3481 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggaattcg
     3541 agctcggtac gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3601 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3661 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3721 caggacagca agggggagga ttgggaagac aatagcccag cttttgttcc ctttagtgag
     3781 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3841 cgctaattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat
     3901 gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc
     3961 atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata
     4021 gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta
     4081 aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc
     4141 atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat
     4201 tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg
     4261 ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc
     4321 ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag atcggccggc
     4381 cgccggcgtc gacgcgcgcg gccgcggcgc gccgttgctg gcgtttttcc ataggctccg
     4441 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4501 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4561 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4621 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4681 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4741 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4801 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4861 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4921 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4981 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5041 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5101 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5161 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5221 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5281 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5341 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5401 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5461 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5521 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5581 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5641 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5701 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5761 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5821 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5881 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5941 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6001 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6061 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6121 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6181 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     6241 tcgtctacgc gcgcgccggc ggcgcgccgt cgacggccgg ccgcggccgc
//
