LOCUS       Exported                6278 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  T7 Expression vector pNam, complete sequence.
ACCESSION   AF147463
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  JOURNAL   Unpublished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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6278
                     /organism="Expression vector pNam"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:101373"
     CDS             complement(144..1004)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /protein_id="AAD49603.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(144..1004)
                     /gene="bla"
                     /label=bla
     promoter        complement(1005..1109)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      1169..1216
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1292..1450)
                     /codon_start=1
                     /gene="cbd"
                     /product="chitin binding domain"
                     /label=cbd
                     /protein_id="AAD49604.1"
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     gene            complement(1292..1450)
                     /gene="cbd"
                     /label=cbd
     CDS             complement(1295..1450)
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             complement(1484..2845)
                     /codon_start=1
                     /gene="int"
                     /product="intein"
                     /label=int
                     /protein_id="AAD49605.1"
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRGNGIRNNLN
                     TENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRETFLAGLIDSDGYVTDEHGIKATIK
                     TIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVTKHKISYAIYMSGGDVLLNVLSKCA
                     GSKKFRPAPAAAFARECRGFYFELQELKEDDYYGITLSDDSDHQFLLGSQVVVHA"
     gene            complement(1484..2845)
                     /gene="int"
                     /label=int
     CDS             complement(1560..1571)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(2027..2845)
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="5 region of the intein from the yeast Vma1 
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 5 region
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     misc_feature    2846..2899
                     /label=multiple cloning site
                     /note="multiple cloning site"
     RBS             2904..2926
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2941..2965
                     /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)."
     promoter        complement(2966..2984)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2969..2984
                     /regulatory_class="promoter"
                     /note="T7 promoter"
     promoter        3297..3374
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3375..4457
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /protein_id="AAD49606.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            3375..4457
                     /gene="lacI"
                     /label=lacI
     misc_feature    4829..6277
                     /label=pSC101 replicon
                     /note="pSC101 replicon"
     rep_origin      4956..5178
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5226..6176
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"