LOCUS       sequence-399567-        5449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET calibration: Dimer construct consisting of mTurquoise2 and 
            mVenus with a 2A peptide linker for posttranslational linker 
            cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5449)
  AUTHORS   Feldmann C, Schanzler M, Ben-Johny M, Wahl-Schott C
  TITLE     Protocol for deriving proximity, affinity, and stoichiometry of 
            protein interactions using image-based quantitative two-hybrid FRET.
  JOURNAL   STAR Protoc. 2023 Jul 28;4(3):102459. doi: 
            10.1016/j.xpro.2023.102459.
  PUBMED    37516972
REFERENCE   2  (bases 1 to 5449)
  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"; journalName: "STAR 
            Protoc."; date: "2023-07-28"; volume: "4(3):102459. doi"; pages: " 
            10.1016/j.xpro.2023.102459"
COMMENT     Sequence Label: Dimer cleaved
FEATURES             Location/Qualifiers
     source          1..5449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             143..937
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             143..937
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(197..216)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     807..826
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1125..1144)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1169..1216
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1545..2133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2034..2053
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2308..2611
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2612..2815
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2766..2786
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(2844..2846,2847..2849,2850..3560)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3567..3620
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      3636..3645
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3642..3644,3645..3647,3648..4361)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    4484..4605
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4521..4540)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4575..4594
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4612..5067)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4749..4770)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4961..4980
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5094..5198
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(5200..5449,1..108)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5200..5391
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5223..5243)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(5408..5449,1..94)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
       61 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
      121 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
      181 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
      241 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
      301 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
      361 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
      421 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
      481 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
      541 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
      601 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
      661 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
      721 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
      781 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
      841 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
      901 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
      961 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     1021 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     1081 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     1141 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     1201 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     1261 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     1321 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     1381 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     1441 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     1501 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1561 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1621 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1681 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1741 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1801 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1861 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1921 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1981 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2041 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2101 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2161 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     2221 gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt
     2281 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     2341 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     2401 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     2461 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     2521 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     2581 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     2641 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     2701 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     2761 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt
     2821 agtgaaccgt cagatccgct agcatggtga gcaagggcga ggagctgttc accggggtgg
     2881 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2941 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gctgatctgc accaccggca
     3001 agctgcccgt gccctggccc accctcgtga ccaccctggg ctacggcctg cagtgcttcg
     3061 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     3121 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     3181 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     3241 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     3301 tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg
     3361 aggacggcgg cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     3421 ccgtgctgct gcccgacaac cactacctga gctaccagtc caagctgagc aaagacccca
     3481 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     3541 gcatggacga gctgtacaag tccggagagg gcagaggaag tcttctaaca tgcggtgacg
     3601 tggaggagaa tcccggccct ggggatccac cggtcgccac catggtgagc aagggcgagg
     3661 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3721 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3781 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgtcct
     3841 ggggcgtgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3901 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3961 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     4021 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactact
     4081 ttagcgacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag gccaacttca
     4141 agatccgcca caacatcgag gacggcggcg tgcagctcgc cgaccactac cagcagaaca
     4201 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca
     4261 agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     4321 ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc gactctagat
     4381 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     4441 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     4501 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4561 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa
     4621 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4681 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4741 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4801 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4861 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4921 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4981 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     5041 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5101 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5161 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     5221 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     5281 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     5341 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     5401 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgccc
//
