LOCUS       Exported                6101 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Phagemid cloning vector pSIT, complete sequence.
ACCESSION   U17139
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6101)
  AUTHORS   Andreansky M, Hunter E.
  TITLE     Phagemid pSIT permits efficient in vitro mutagenesis and tightly 
            controlled expression in E. coli
  JOURNAL   BioTechniques 16 (4), 626-628 (1994)
  PUBMED    8024782
REFERENCE   2  (bases 1 to 6101)
  AUTHORS   Promega Corporation.
  TITLE     Altered Sites in vitro Mutagenesis System Technical Manual
  JOURNAL   (in) Editor,N.A. (Ed.); TECHNICAL MANUAL, Vol. 1: 1-22; Promega 
            Corporation, Madison, WI (1990)
REFERENCE   3  (bases 1 to 6101)
  AUTHORS   Promega Corporation.
  TITLE     Altered Sites II in vitro Mutagenesis System Technical Manual
  JOURNAL   (in) Editor,N.A. (Ed.); TECHNICAL MANUAL, Vol. 2: 1-33; Promega 
            Corporation, Madison, WI (1994)
REFERENCE   4  (bases 1 to 6101)
  AUTHORS   Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW.
  TITLE     Use of T7 RNA polymerase to direct expression of cloned genes
  JOURNAL   Meth. Enzymol. 185, 60-89 (1990)
  PUBMED    2199796
REFERENCE   5  (bases 1 to 6101)
  AUTHORS   Hunter E.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-NOV-1994) Eric Hunter, Microbiology, University of 
            Alabama at Birmingham, 845 19th St.S., Bevill Biomedical Research 
            Building, Rm 256, Birmingham, AL 35294, USA
REFERENCE   6  (bases 1 to 6101)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6101
                     /organism="Cloning vector pSIT"
                     /lab_host="Escherichia coli"
                     /mol_type="genomic DNA"
                     /note="pSIT is a phagemid that permits both effective 
                     mutagenesis and tightly controlled high-level expression of
                     proteins from a single construct, without the need for 
                     subcloning every mutant from mutagenesis to expression 
                     vector"
                     /db_xref="taxon:37979"
     regulatory      19..36
                     /regulatory_class="promoter"
                     /note="phage T7 gene 10 promoter, the strongest phage T7 
                     promoter, recognized by phage T7 RNA polymerase"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    39..63
                     /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)."
     protein_bind    43..59
                     /function="binds lac repressor (product of the lacIq gene) 
                     and thus regulates the upstream phage T7 gene 10 promoter"
                     /label=lac repressor binding site
                     /bound_moiety="lac repressor"
     RBS             78..100
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     regulatory      94..99
                     /regulatory_class="ribosome_binding_site"
     CDS             107..208
                     /codon_start=1
                     /note="33 amino acid polypeptide; NcoI site (105) allows 
                     cloning at translation initiation (ATG); BamHI (141) allows
                     cloning at 12 and EspI (193) at 30 amino acids downstream 
                     from ATG"
                     /protein_id="AAA56766.1"
                     /translation="MASMTGGQQMGRIRLLTKPERKLSWLLPPLSNN"
     CDS             107..139
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     regulatory      149..252
                     /regulatory_class="terminator"
                     /note="phage T7 transcription terminator"
     terminator      205..252
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        473..501
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             549..1739
                     /codon_start=1
                     /product="tetracycline resistance protein"
                     /label=tetracycline resistance protein
                     /note="396 aa protein that confers resistance to 
                     tetracycline; initiation of transcription is about 35 
                     nucleotides upstream of the translation initiation"
                     /protein_id="AAA56767.1"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     1957..1973
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1989..2005)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2013..2029
                     /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(2037..2067)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2082..2103
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(2116..3198)
                     /codon_start=1
                     /gene="lacIq"
                     /product="lac operon repressor"
                     /label=lacIq
                     /note="360 aa protein that binds to lac operator sequence; 
                     According to Pharmacia (lacIq gene from pGEX-3), there is a
                     HpaI at the 3 terminus of lacIq gene, yet the GTTAAC is 
                     not in the sequence. However, HpaI does linearize pSIT at 
                     around nucleotide 2200"
                     /protein_id="AAA56768.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(2116..3198)
                     /gene="lacIq"
                     /label=lacIq
     promoter        complement(3199..3276)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     rep_origin      3518
                     /note="ColE1 based origin of DNA replication, that proceeds
                     counterclockwise from T (nucleotide 3518); ROP that down 
                     regulates plasmid copy number has been deleted in pSIT, 
                     providing higher DNA yields"
     rep_origin      complement(3520..4108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4563..5135)
                     /codon_start=1
                     /note="truncated beta-lactamase gene with a 4bp deletion, 
                     repaired by mutagenesis with oligo 
                     GTTGCCATTGCTGCAGGCATCGTGGTG (nucleotides 4573-4595) 
                     restoring ampicillin resistance and PstI site by 4 bp 
                     insertion"
                     /protein_id="AAA56769.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPQWQQRCANY"
     promoter        complement(5136..5240)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5576..6031)
                     /direction=LEFT
                     /note="phage f1 replication origin, allows procuring single
                     stranded DNA from pSIT upon infection with a helper phage 
                     M13KO7 or R408"