LOCUS       sequence-249293-        6476 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 6476)
  TITLE     Lewin Lab Plasmids
REFERENCE   2  (bases 1 to 6476)
  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-472    N-Tag UnaG[1] L20 FRB EBPF2
FEATURES             Location/Qualifiers
     source          1..6476
                     /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             1343..1618
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS"
     misc_feature    join(1635..2209,2210..2212,2213..2221)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1802..1819)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2029..2048
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2222..2224,2225..2227,2228..2941)
                     /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(2267..2288)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     intron          2987..3119
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_feature    3128..3716
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3181..3201)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3599..3610)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3743..3760)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3749..3860
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3956..3976)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3956..3974)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3995..4011)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3995..4011)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4008..4030)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(4113..4132)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4178..4233
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4232..4251)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     rep_origin      complement(4612..5200)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4692..4711)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5371..6162,6163..6231))
                     /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     5994..6013
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6232..6336)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6404..6422
                     /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 caccatggtc gaaaagttcg tgggcacatg gaagattgcc gatagccaca acttcggcga
     1081 atacctcaaa gctatagggg caccaaaaga gcttagcgat ggtggagatg cgacaactcc
     1141 cacactgtac attagccaga aagacggaga caagatgact gtcaagatcg agaatgggcc
     1201 tcccaccttt ctggacacac aggttaagtt caaactgggt gaggagtttg acgagtttcc
     1261 cagtgatcgc aggaaaggga gtcctggcgg tggatccgga ggctctggga gctctgccag
     1321 cggtggcagc acctccgtgg ccatcctctg gcatgagatg tggcatgaag gcctggaaga
     1381 ggcatctcgt ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg tgctggagcc
     1441 cttgcatgct atgatggaac ggggccccca gactctgaag gaaacatcct ttaatcaggc
     1501 ctatggtcga gatttaatgg aggcccaaga gtggtgcagg aagtacatga aatcagggaa
     1561 tgtcaaggac ctcacccaag cctgggacct ctattatcat gtgttccgac gaatctcata
     1621 agaattcaga tccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg
     1681 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc
     1741 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc
     1801 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggta
     1861 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct
     1921 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca
     1981 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca
     2041 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat
     2101 ctggggcctc ggtacacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc
     2161 cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac
     2221 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2281 cggcgacgta aacggccaca agttcagcgt gaggggcgag ggcgagggcg atgccaccaa
     2341 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2401 cctcgtgacc accctgagcc acggcgtgca gtgcttcgcc cgctaccccg accacatgaa
     2461 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2521 cttcaaggac gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2581 ggtgaaccgc atcgagctga agggcgtcga cttcaaggag gacggcaaca tcctggggca
     2641 caagctggag tacaacttca acagccacaa catctatatc atggccgtca agcagaagaa
     2701 cggcatcaag gtgaacttca agatccgcca caacgtggag gacggcagcg tgcagctcgc
     2761 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacagcca
     2821 ctacctgagc acccagtccg tgctgagcaa agaccccaac gagaagcgcg atcacatggt
     2881 cctgctggag ttccgcaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     2941 aaccggtgct cgaggtaccg atgaatagct aaggtagagg ccgcaggtaa gtatcaaggt
     3001 tacaagacag gtttaaggag accaatagaa actgggcttg tcgagacaga gaagactctt
     3061 gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc tctccacagg
     3121 tgtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3181 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3241 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3301 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3361 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3421 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3481 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
     3541 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3601 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3661 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg
     3721 aattcgagct cggtacgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3781 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3841 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3901 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcccagcttt tgttcccttt
     3961 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4021 gttatccgct aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg
     4081 gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg
     4141 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     4201 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     4261 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg
     4321 gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct
     4381 gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt
     4441 tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga
     4501 tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcg
     4561 gccggccgcc ggcgtcgacg cgcgcggccg cggcgcgccg ttgctggcgt ttttccatag
     4621 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4681 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4741 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4801 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4861 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4921 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4981 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5041 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5101 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5161 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5221 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5281 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5341 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5401 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5461 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5521 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5581 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5641 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5701 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5761 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5821 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5881 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5941 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6001 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6061 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6121 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6181 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6241 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6301 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6361 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     6421 gccctttcgt ctacgcgcgc gccggcggcg cgccgtcgac ggccggccgc ggccgc
//
