LOCUS       sequence-171300-        6148 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 6148)
  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 6148)
  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..6148
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             95..346
                     /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     283..304
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        458..476
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    477..501
                     /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(565..567,568..570,571..1029)
                     /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(610..631)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(871..890)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1094..1138
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1176..1194)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1190..1237
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1274..1729
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1361..1380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1571..1592
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1844..2635,2636..2704))
                     /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     2467..2486
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2705..2797)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2878..3466
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3367..3386
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3620..3637
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3652..3794
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3720..3739)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3880..3902
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3896..4087)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4659..5741)
                     /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     5703..5722
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5742..5819)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6025..6044
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6132..6148,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggaattc attcgcgagg atccgacaag cagaagaacg gcatcaaggc
      121 caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca
      181 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta
      241 ccagtccaaa ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
      301 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gatccgaatt
      361 cgagctcggc gcgcctgcag gtcgacaagc ttgcggccgc ataatgctta agtcgaacag
      421 aaagtaatcg tattgtacac ggccgcataa tcgaaattaa tacgactcac tataggggaa
      481 ttgtgagcgg ataacaattc cccatcttag tatattagtt aagtataaga aggagatata
      541 catatggaat tcgctagcag atctatggtg agcaagggcg aggagctgtt caccggggtg
      601 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
      661 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agctgatctg caccaccggc
      721 aagctgcccg tgccctggcc caccctcgtg accaccctgg gctacggcct gcagtgcttc
      781 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
      841 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
      901 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
      961 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1021 ctcaccgcct gaggatctca attggatatc ggccggccac gcgatcgctg acgtcggtac
     1081 cctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc
     1141 tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc
     1201 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1261 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1321 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1381 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1441 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1501 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1561 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1621 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1681 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc tggcggcacg
     1741 atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     1801 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     1861 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     1921 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     1981 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     2041 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     2101 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     2161 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     2221 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     2281 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     2341 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     2401 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     2461 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     2521 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     2581 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     2641 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     2701 tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg tctcatgagc
     2761 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca aaatccctta
     2821 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2881 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2941 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3001 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3061 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3121 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3181 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3241 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3301 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3361 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3421 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3481 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3541 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3601 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3661 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt
     3721 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3781 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3841 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3901 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3961 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4021 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4081 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4141 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4201 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4261 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4321 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4381 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4441 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4501 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg
     4561 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4621 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4681 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4741 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4801 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4861 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4921 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4981 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5041 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5101 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5161 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5221 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5281 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5341 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5401 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5461 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5521 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5581 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5641 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5701 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5761 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5821 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     5881 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     5941 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     6001 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     6061 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga
     6121 tcccgcgaaa ttaatacgac tcactata
//
