LOCUS       sequence-270714-        6183 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 6183)
  TITLE     Lewin Lab Plasmids
REFERENCE   2  (bases 1 to 6183)
  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-145    N-Tag GFP[2] L32 GCNZipper EBPF2
FEATURES             Location/Qualifiers
     source          1..6183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        151..530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        531..734
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     685..705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     731..755
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          870..1002
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1017..1026
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1254..1310
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     misc_feature    join(1342..1916,1917..1919,1920..1928)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1509..1526)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1736..1755
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1929..1931,1932..1934,1935..2648)
                     /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(1974..1995)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     intron          2694..2826
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_feature    2835..3423
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2888..2908)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3306..3317)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3450..3467)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3456..3567
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3663..3683)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3663..3681)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3702..3718)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3702..3718)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3715..3737)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(3820..3839)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3885..3940
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3939..3958)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     rep_origin      complement(4319..4907)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4399..4418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5078..5869,5870..5938))
                     /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     5701..5720
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5939..6043)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6111..6129
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctgcctccag gcctcaatat tggccattag ccatattatt cattggttat atagcataaa
       61 tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg tacatttata
      121 ttggctcatg tccaatatga ccgccatgtt ggcattgatt attgactagt tattaatagt
      181 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      241 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      301 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      361 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta
      421 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
      481 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      541 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      601 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      661 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      721 atataagcag agctcgttta gtgaaccgtc agatcactag aagctttatt gcggtagttt
      781 atcacagtta aattgctaac gcagtcagtg cttctgacac aacagtctcg aacttaagct
      841 gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag
      901 gagaccaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac
      961 ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac tcccaggcca
     1021 ccatggaaaa gagggaccac atggtgctgc tcgagtacgt cacagcagct ggcatcaccg
     1081 atgccagcga cgtgggtggc ggagggagcg aaggcggcgg atccggaggg cctggctctg
     1141 gcggagaggg gagcgctgga ggcgggtcag ccggtggtgg gagtaacact gaagccgcca
     1201 ggcgttctcg tgcgagaaag ttgcaaagaa tgaaacaact tgaagacaag gttgaagaat
     1261 tgctttcgaa aaattatcac ttggaaaatg aggttgccag attaaagaaa ttagttggcg
     1321 aacgctaaga attcagatcc gcccctctcc ctcccccccc cctaacgtta ctggccgaag
     1381 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     1441 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     1501 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     1561 tctggtagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     1621 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     1681 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     1741 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     1801 atctgatctg gggcctcggt acacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
     1861 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     1921 ccacaaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1981 agctggacgg cgacgtaaac ggccacaagt tcagcgtgag gggcgagggc gagggcgatg
     2041 ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2101 ggcccaccct cgtgaccacc ctgagccacg gcgtgcagtg cttcgcccgc taccccgacc
     2161 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2221 ccatcttctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg
     2281 acaccctggt gaaccgcatc gagctgaagg gcgtcgactt caaggaggac ggcaacatcc
     2341 tggggcacaa gctggagtac aacttcaaca gccacaacat ctatatcatg gccgtcaagc
     2401 agaagaacgg catcaaggtg aacttcaaga tccgccacaa cgtggaggac ggcagcgtgc
     2461 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2521 acagccacta cctgagcacc cagtccgtgc tgagcaaaga ccccaacgag aagcgcgatc
     2581 acatggtcct gctggagttc cgcaccgccg ccgggatcac tctcggcatg gacgagctgt
     2641 acaagtaaac cggtgctcga ggtaccgatg aatagctaag gtcgaggccg caggtaagta
     2701 tcaaggttac aagacaggtt taaggagacc aatagaaact gggcttgtcg agacagagaa
     2761 gactcttgcg tttctgatag gcacctattg gtcttactga catccacttt gcctttctct
     2821 ccacaggtgt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     2881 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     2941 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     3001 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     3061 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     3121 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     3181 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct
     3241 ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     3301 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     3361 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     3421 cgcctggaat tcgagctcgg tacgatcagc ctcgactgtg ccttctagtt gccagccatc
     3481 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3541 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3601 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagcc cagcttttgt
     3661 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     3721 tgaaattgtt atccgctaat tcactcctca ggtgcaggct gcctatcaga aggtggtggc
     3781 tggtgtggcc aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa
     3841 ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat
     3901 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag
     3961 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca
     4021 tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc
     4081 ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat
     4141 attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact
     4201 agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg
     4261 gagatcggcc ggccgccggc gtcgacgcgc gcggccgcgg cgcgccgttg ctggcgtttt
     4321 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4381 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4441 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4501 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4561 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4621 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4681 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4741 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4801 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4861 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4921 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4981 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5041 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5101 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5161 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5221 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5281 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5341 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5401 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5461 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5521 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5581 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5641 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5701 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5761 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5821 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5881 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5941 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6001 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6061 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     6121 tcacgaggcc ctttcgtcta cgcgcgcgcc ggcggcgcgc cgtcgacggc cggccgcggc
     6181 cgc
//
