LOCUS       Exported               11340 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses camelid anti-GFP nanobody fused to TRPV1 and GFP-ferritin 
            chimera fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    pVQ CMV NanoV1-2a-EGFP ferritin
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11340)
  AUTHORS   Stanley SA, Kelly L, Latcha KN, Schmidt SF, Yu X, Nectow AR, Sauer 
            J, Dyke JP, Dordick JS, Friedman JM
  TITLE     Bidirectional electromagnetic control of the hypothalamus regulates 
            feeding and metabolism.
  JOURNAL   Nature. 2016 Mar 31;531(7596):647-50. doi: 10.1038/nature17183. Epub
            2016 Mar 23.
  PUBMED    27007848
REFERENCE   2  (bases 1 to 11340)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature17183"; journalName: "Nature"; date: "2016-03-31- 
            31"; volume: "531"; issue: "7596"; pages: "647-50"
FEATURES             Location/Qualifiers
     source          1..11340
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     389..411
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     polyA_signal    554..688
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(591..610)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     645..664
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(1328..1351)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     RBS             1506..1514
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3 end of the 16S rRNA (Chen et al., 1994)"
     CDS             complement(1928..2644)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(1928..2644)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1970..1991)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2319..2338
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2578..2599
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2660..2713)
                     /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"
     CDS             complement(5258..5599)
                     /codon_start=1
                     /product="GFP-binding fragment of a single-chain camelid 
                     antibody (Rothbauer et al., 2008)"
                     /label=GFP nanobody
                     /translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
                     WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYSNVNVGFEYW
                     GQGTQVTVSS"
     regulatory      5605..5614
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(5643..5667)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    5694..5727
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        complement(5775..5978)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5804..5824)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5979..6282
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    complement(6372..6392)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(6418..6434)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6419..6435)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     misc_signal     6476..6626
                     /label=Ad5 Psi
                     /note="packaging signal for adenovirus serotype 5"
     repeat_region   6714..6816
                     /label=ITR
                     /note="inverted terminal repeat of human adenovirus 
                     serotype 5"
     primer_bind     complement(6844..6862)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6930..7034
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7035..7895
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7253..7272)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8066..8654
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8555..8574
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"