LOCUS       sequence-249294-        6512 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 6512)
  TITLE     Lewin Lab Plasmids
REFERENCE   2  (bases 1 to 6512)
  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-473    C-Tag UnaG[1] L32 FRB EBPF2
FEATURES             Location/Qualifiers
     source          1..6512
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        152..531
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        532..735
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     686..706
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     732..756
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          871..1003
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1018..1027
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1022..1031
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1033..1308
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS"
     misc_feature    join(1670..2244,2245..2247,2248..2256)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1837..1854)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2064..2083
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2257..2259,2260..2262,2263..2976)
                     /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(2302..2323)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     intron          3022..3154
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_feature    3163..3751
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3216..3236)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3634..3645)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3778..3795)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3784..3895
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3991..4011)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3991..4009)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4030..4046)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4030..4046)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4043..4065)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(4148..4167)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4213..4268
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4267..4286)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     rep_origin      complement(4647..5235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4727..4746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5406..6197,6198..6266))
                     /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     6029..6048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6267..6371)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6439..6457
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cctgcctcca ggcctcaata ttggccatta gccatattat tcattggtta tatagcataa
       61 atcaatattg gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat
      121 attggctcat gtccaatatg accgccatgt tggcattgat tattgactag ttattaatag
      181 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      241 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      301 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      361 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct
      421 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg
      481 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      541 ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      601 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      661 tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      721 tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt
      781 tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc
      841 tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa
      901 ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca
      961 cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccaggcc
     1021 accatggtgg ccatcctctg gcatgagatg tggcatgaag gcctggaaga ggcatctcgt
     1081 ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg tgctggagcc cttgcatgct
     1141 atgatggaac ggggccccca gactctgaag gaaacatcct ttaatcaggc ctatggtcga
     1201 gatttaatgg aggcccaaga gtggtgcagg aagtacatga aatcagggaa tgtcaaggac
     1261 ctcacccaag cctgggacct ctattatcat gtgttccgac gaatctcaga cgtgggtggc
     1321 ggagggagcg aaggcggcgg atccggaggg cctggctctg gcggagaggg gagcgctgga
     1381 ggcgggtcag ccggtggtgg gagtgtcgaa aagttcgtgg gcacatggaa gattgccgat
     1441 agccacaact tcggcgaata cctcaaagct ataggggcac caaaagagct tagcgatggt
     1501 ggagatgcga caactcccac actgtacatt agccagaaag acggagacaa gatgactgtc
     1561 aagatcgaga atgggcctcc cacctttctg gacacacagg ttaagttcaa actgggtgag
     1621 gagtttgacg agtttcccag tgatcgcagg aaataagaat tcagatccgc ccctctccct
     1681 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     1741 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     1801 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     1861 tgttgaatgt cgtgaaggaa gcagttcctc tggtagcttc ttgaagacaa acaacgtctg
     1921 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     1981 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     2041 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     2101 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtac acatgcttta
     2161 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     2221 tcctttgaaa aacacgatga taatatggcc acaaccatgg tgagcaaggg cgaggagctg
     2281 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2341 agcgtgaggg gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc
     2401 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gagccacggc
     2461 gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2521 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg cacctacaag
     2581 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2641 gtcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc
     2701 cacaacatct atatcatggc cgtcaagcag aagaacggca tcaaggtgaa cttcaagatc
     2761 cgccacaacg tggaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     2821 atcggcgacg gccccgtgct gctgcccgac agccactacc tgagcaccca gtccgtgctg
     2881 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttccg caccgccgcc
     2941 gggatcactc tcggcatgga cgagctgtac aagtaaaccg gtgctcgagg taccgatgaa
     3001 tagctaaggt cgaggccgca ggtaagtatc aaggttacaa gacaggttta aggagaccaa
     3061 tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc acctattggt
     3121 cttactgaca tccactttgc ctttctctcc acaggtgtcg acaatcaacc tctggattac
     3181 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3241 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3301 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3361 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3421 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3481 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3541 gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg
     3601 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3661 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3721 agtcggatct ccctttgggc cgcctccccg cctggaattc gagctcggta cgatcagcct
     3781 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3841 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3901 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3961 attgggaaga caatagccca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     4021 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctaattc actcctcagg
     4081 tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc
     4141 actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc
     4201 tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt
     4261 gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt
     4321 tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat
     4381 aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag
     4441 ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat
     4501 ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc
     4561 cagtcatagc tgtccctctt ctcttatgga gatcggccgg ccgccggcgt cgacgcgcgc
     4621 ggccgcggcg cgccgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5281 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5341 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5401 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5461 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5521 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5581 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5641 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5701 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5761 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5821 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5881 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5941 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6001 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6061 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6121 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6181 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6241 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6301 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6361 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6421 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctacg cgcgcgccgg
     6481 cggcgcgccg tcgacggccg gccgcggccg cg
//
