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L4440 vector (V012784#)

Basic Vector Information

E. coli bacteria carrying RNAi plasmids that produce the desired dsRNA can be fed to worms, and the dsRNA is transferred to the worm via the intestinal tract. RNAi plasmids typically consist of DNA coding sequence from the intended target gene cloned between two T7lac promoters. The plasmid also has a selectable marker that confers resistance to an antibiotic, in this case ampicillin. In 7.15, you will use the E. coli strain HT115 carrying various L4440 plasmids, each containing a different cloned gene sequence. HT115 is an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity. To induce dsRNA production from these plasmids, the HT115 bacteria is grown on special RNAi NGM feeding plates that contain IPTG and the ampicillin analog carbenicillin. Carbenicillin is preferred over ampicillin because it tends to be more stable.

Basic Vector Information
Vector Name L4440 Antibiotic Resistance Ampicillin
Length 2790 bp Type RNAi
Source Andrew Fire Copy Number High copy number
Promoter T7 Cloning Method Enzyme Cut
Expression Method IPTG induced

L4440 vector Vector Map

T7 promoter SK primer KS primer T7 promoter M13 fwd f1 ori bla bla ori

L4440 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                2790 bp ds-DNA     circular SYN 23-SEP-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    L4440
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2790)
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..2790
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        19..37
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     95..111
                     /note="SK primer"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(181..197)
                     /note="KS primer"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(223..241)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(251..267)
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      409..864
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        890..994
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             995..1855
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2026..2614
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"

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