pNam vector (V004362)

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LOCUS       40924_32988        6278 bp DNA     circular SYN 18-DEC-2018
DEFINITION  T7 Expression vector pNam, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6278)
  AUTHORS   Hoang TT, Stern RJ, McNeil MR, Schweizer HP.
  TITLE     Construction and use of low-copy number T7 expression vectors for 
            purification of problem proteins: affinity purification of 
            Mycobacterium tuberculosis RmlD and Pseudomonas aeruginosa LasI and 
            RhlI proteins, and functional analysis of RhlI
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6278)
  AUTHORS   Hoang TT, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (30-APR-1999) Microbiology, Colorado State University, 
            Fort Collins, CO 80523, USA
REFERENCE   3  (bases 1 to 6278)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6278)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (30-APR-1999) Microbiology, Colorado State University, Fort Collins,
            CO 80523, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(147..1004)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1005..1109)
                     /label=AmpR promoter
     terminator      complement(1169..1216)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1295..1450)
                     /codon_start=1
                     /label=CBD
                     /note="chitin binding domain from chitinase A1 (Watanabe et
                     al., 1994)"
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             complement(1490..2023)
                     /codon_start=1
                     /label=Sce VMA intein 3' region
                     /note="modified 3' region of the intein from the yeast Vma1
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /translation="GIRNNLNTENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRETFL
                     AGLIDSDGYVTDEHGIKATIKTIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVTKHK
                     ISYAIYMSGGDVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKEDDYYGITLS
                     DDSDHQFLLGSQVVV"
     CDS             complement(2027..2845)
                     /codon_start=1
                     /label=Sce VMA intein 5' region
                     /note="5' region of the intein from the yeast Vma1 subunit
                     of the vacuolar ATPase (Chong et al., 1998)"
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     misc_feature    2846..2899
                     /label=multiple cloning site
                     /note="multiple cloning site"
     RBS             complement(2904..2926)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(2941..2965)
                     /label=lac operator
                     /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)."
     promoter        complement(2966..2984)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3297..3374
                     /label=lacI promoter
     CDS             3375..4454
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    4470..4491
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      4956..5178
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5226..6173
                     /codon_start=1
                     /label=Rep101
                     /note="RepA protein needed for replication with the pSC101 
                     origin"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"