LOCUS       sequence-364922-        6538 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-Leucine 
            Zipper-amino-half_mVenus BIFC fragment n and Vesicle Nucleating 
            peptide-Leucine Zipper-carboxyl-half_mVenus BIFC fragment fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6538)
  AUTHORS   Eastwood TA, Baker K, Streather BR, Allen N, Wang L, Botchway SW, 
            Brown IR, Hiscock JR, Lennon C, Mulvihill DP
  TITLE     High-yield vesicle-packaged recombinant protein production from E. 
            coli.
  JOURNAL   Cell Rep Methods. 2023 Feb 2;3(2):100396. doi: 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27.
  PUBMED    36936078
REFERENCE   2  (bases 1 to 6538)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep 
            Methods."; date: "2023-02-2"; volume: "3(2):100396. doi"; pages: " 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27"
COMMENT     Sequence Label: pETDuet-1_VNp-LZ-VenusN154_VNp-LZ-VenusC155 (B...
FEATURES             Location/Qualifiers
     source          1..6538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     513..532
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(552..629)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     835..854
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        942..960
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    961..985
                     /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             1000..1022
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1176..1232
                     /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"
     CDS             1248..1499
                     /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     1436..1457
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        1611..1629
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1630..1654
                     /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             1844..1900
                     /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"
     CDS             join(1916..1918,1919..1921,1922..2380)
                     /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(1961..1982)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2222..2241)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2445..2489
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2527..2545)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2541..2588
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2625..3080
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2712..2731)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2922..2943
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3195..3986,3987..4055))
                     /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     3818..3837
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4056..4148)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4229..4817
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4718..4737
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4971..4988
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5003..5142
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5068..5087)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5228..5250
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5244..5435)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6007..6538,1..551))
                     /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"
ORIGIN
        1 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
       61 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
      121 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
      181 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
      241 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
      301 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
      361 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
      421 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
      481 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
      541 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
      601 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
      661 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
      721 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
      781 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
      841 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
      901 tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg
      961 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     1021 gatataccat ggatgtattc atgaaaggac tttcaaaggc caaggaggga gttgtggcag
     1081 ctgctgagaa aaccaaacag ggtgtggcag aagcagcagg aaagacaaaa gagggtgttc
     1141 tcagatcacg tatgaaacag ctggaagata aagttgaaga actgctgagc aaaaactacc
     1201 acctggaaaa tgaagttgcc cgtctgaaaa aactggttgg tggatccgac aagcagaaga
     1261 acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg
     1321 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1381 actacctgag ctaccagtcc aaactgagca aagaccccaa cgagaagcgc gatcacatgg
     1441 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1501 aaagatccga attcgagctc ggcgcgcctg caggtcgaca agcttgcggc cgcataatgc
     1561 ttaagtcgaa cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact
     1621 cactataggg gaattgtgag cggataacaa ttccccatct tagtatatta gttaagtata
     1681 agaaggagat atacatatgg atgtattcat gaaaggactt tcaaaggcca aggagggagt
     1741 tgtggcagct gctgagaaaa ccaaacaggg tgtggcagaa gcagcaggaa agacaaaaga
     1801 gggtgttctc agatcacgta tgaaacagct ggaagataaa gttgaagaac tgctgagcaa
     1861 aaactaccac ctggaaaatg aagttgcccg tctgaaaaaa ctggttggta gatctatggt
     1921 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1981 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2041 gctgaccctg aagctgatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2101 gaccaccctg ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     2161 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2221 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2281 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2341 ggagtacaac tacaacagcc acaacgtcta tctcaccgcc tgaggatctc aattggatat
     2401 cggccggcca cgcgatcgct gacgtcggta ccctcgagtc tggtaaagaa accgctgctg
     2461 cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg
     2521 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     2581 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     2641 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2701 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2761 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2821 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2881 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2941 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3001 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3061 aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa aaggatcttc
     3121 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3181 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3241 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3301 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3361 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3421 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3481 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3541 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3601 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3661 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3721 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3781 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3841 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3901 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3961 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4021 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atcatgattg
     4081 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4141 taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4201 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4261 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4321 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4381 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4441 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4501 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4561 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4621 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4681 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4741 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4801 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4861 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4921 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4981 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5041 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5101 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     5161 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5221 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     5281 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     5341 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     5401 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     5461 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     5521 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     5581 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     5641 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     5701 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     5761 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     5821 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     5881 agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5941 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     6001 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6061 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     6121 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     6181 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     6241 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac
     6301 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6361 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6421 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6481 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaac
//
