LOCUS       sequence-399579-        6169 bp    DNA     circular SYN 15-APR-2025
DEFINITION  FRET acceptor: Mutated IQ6 domain of Moysin Va tagged with 
            N-terminal mVenus (lower binding affinity).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6169)
  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 6169)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: mVenus-IQ6 R6A
FEATURES             Location/Qualifiers
     source          1..6169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(901..903,904..906,907..1617)
                     /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"
     primer_bind     complement(946..967)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1207..1226)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1554..1575
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1760..1777)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1766..1990
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2036..2464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2123..2142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2333..2354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2473..2493)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2478..2807
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2658..2793
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2720..2739
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2874..3668
                     /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="MIEQDGLHAGSPDAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2928..2947)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3538..3557
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3842..3963
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3879..3898)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3933..3952
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4012..4028)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4012..4028)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4025..4047)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4036..4052
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4060..4066,4067..4084,4085..4090))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4105..4126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4243..4260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4414..5002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4494..4513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5173..5964,5965..6033))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSHATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5796..5815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6034..6138)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      961 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1021 ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1081 ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
     1141 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1201 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1261 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1321 aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac
     1381 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc
     1441 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1501 tacctgagct accagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1561 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggga
     1621 tccaccatga aagccatcgt ctaccttcag tgctgcttcg cacggatgat ggccaagcgt
     1681 gagctgaaga aactcaaaat tgaggctcgc tctgtggaac gctacaagaa gtaagggccc
     1741 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1801 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1861 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     1921 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     1981 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2041 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2101 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2161 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2221 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2281 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2341 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2401 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2461 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2521 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2581 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2641 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2701 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     2761 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     2821 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     2881 aacaagatgg attgcacgca ggttctccgg acgcttgggt ggagaggcta ttcggctatg
     2941 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3001 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3061 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3121 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3181 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3241 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3301 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3361 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3421 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3481 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3541 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3601 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3661 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     3721 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     3781 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     3841 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3901 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3961 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4021 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4081 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4141 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4201 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4261 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4321 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4381 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4441 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4501 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4561 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4621 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4681 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4741 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4801 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4861 atttggtatc tgcgctctgc tgaagcaagt taccttcgga aaaagagttg gtagctcttg
     4921 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4981 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5041 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5101 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5161 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5221 tcgttcatcc atagttgcat gactccccgt cgtgtagata actacgatac gggagggctt
     5281 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5341 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5401 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5461 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5521 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5581 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5641 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5701 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5761 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5821 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5881 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5941 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6001 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6061 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6121 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
