LOCUS       sequence-175019-        6145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pETDUET-1 based vector for use in BiMolecular Fluorescence 
            Complementation based assays. MCS at 5' of each BiFC fragment..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6145)
  AUTHORS   Eastwood T, Baker K, Brooker H, Frank S, Mulvihill DP
  TITLE     An enhanced recombinant amino-terminal acetylation system and novel 
            in vivo high-throughput screen for molecules affecting 
            alpha-synuclein oligomerisation.
  JOURNAL   FEBS Lett. 2017 Feb 18. doi: 10.1002/1873-3468.12597.
  PUBMED    28214355
REFERENCE   2  (bases 1 to 6145)
  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: "FEBS Lett. 
            2017 Feb 18. doi: 10.1002/1873-3468.12597."
COMMENT     Sequence Label: pET-BiFC
FEATURES             Location/Qualifiers
     source          1..6145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             25..276
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     213..234
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        388..406
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    407..431
                     /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)."
     CDS             join(495..497,498..500,501..959)
                     /codon_start=1
                     /product="improved N-terminal fragment of mVenus for use in
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN155(I152L)
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYLTA"
     primer_bind     complement(540..561)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(801..820)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1024..1068
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1106..1124)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1120..1167
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1204..1659
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1291..1310)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1501..1522
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1774..2565,2566..2634))
                     /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     2397..2416
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2635..2727)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2808..3396
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3297..3316
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3550..3567
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3582..3721
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3647..3666)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3807..3829
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3823..4014)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4586..5668)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5630..5649
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5669..5746)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5952..5971
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6059..6077
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6078..6102
                     /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)."
     RBS             6117..6139
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgggaattc attcgcgagg atccgacaag cagaagaacg gcatcaaggc caacttcaag
       61 atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc
      121 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccaaa
      181 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
      241 gccgggatca ctctcggcat ggacgagctg tacaagtaaa gatccgaatt cgagctcggc
      301 gcgcctgcag gtcgacaagc ttgcggccgc ataatgctta agtcgaacag aaagtaatcg
      361 tattgtacac ggccgcataa tcgaaattaa tacgactcac tataggggaa ttgtgagcgg
      421 ataacaattc cccatcttag tatattagtt aagtataaga aggagatata catatggaat
      481 tcgctagcag atctatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      541 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      601 gcgatgccac ctacggcaag ctgaccctga agctgatctg caccaccggc aagctgcccg
      661 tgccctggcc caccctcgtg accaccctgg gctacggcct gcagtgcttc gcccgctacc
      721 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      781 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      841 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      901 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat ctcaccgcct
      961 gaggatctca attggatatc ggccggccac gcgatcgctg acgtcggtac cctcgagtct
     1021 ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca
     1081 gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1141 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1201 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1261 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1321 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1381 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1441 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1501 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1561 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1621 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc tggcggcacg atggcatgag
     1681 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1741 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1801 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1861 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     1921 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1981 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2041 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2101 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2161 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2221 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2281 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2341 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     2401 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     2461 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     2521 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2581 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2641 cctttttcaa tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat
     2701 ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt
     2761 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2821 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2881 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2941 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3001 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3061 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3121 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3181 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3241 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3301 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3361 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3421 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3481 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3541 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3601 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3661 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3721 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3781 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3841 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3901 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3961 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4021 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     4081 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4141 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4201 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4261 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4321 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4381 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4441 ccgggtcctc aacgacagga gcacgatcat gctagtcatg ccccgcgccc accggaagga
     4501 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4561 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4621 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4681 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4741 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4801 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4861 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     4921 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     4981 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5041 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5101 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5161 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5221 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5281 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5341 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5401 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5461 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5521 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5581 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5641 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5701 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5761 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     5821 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     5881 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     5941 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     6001 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta
     6061 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     6121 tttaacttta agaaggagat atacc
//
