LOCUS       40924_40492        6101 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Phagemid cloning vector pSIT, complete sequence.
ACCESSION   .
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)
  TITLE     Direct Submission
REFERENCE   7  (bases 1 to 6101)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "BioTechniques"; date: "1994"; volume: "16"; issue: "4"; pages: 
            "626-628"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "(in) 
            Editor,N.A. (Ed.)"; volume: " TECHNICAL MANUAL, Vol. 1"; pages: " 
            1-22; Promega Corporation, Madison, WI (1990"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "(in) 
            Editor,N.A. (Ed.)"; volume: " TECHNICAL MANUAL, Vol. 2"; pages: " 
            1-33; Promega Corporation, Madison, WI (1994"
COMMENT     SGRef: number: 4; type: "Journal Article"; journalName: "Meth. 
            Enzymol. 185, 60-89 (1990)"
COMMENT     SGRef: number: 5; type: "Journal Article"; journalName: "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"
COMMENT     SGRef: number: 6; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    39..63
                     /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)."
     RBS             78..100
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             107..139
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
     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..1736
                     /label=TcR
                     /note="tetracycline efflux protein"
     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    complement(2013..2029)
                     /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(2037..2067)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2082..2103)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(2119..3198)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(3199..3276)
                     /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"
     misc_feature    complement(4577..4951)
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene;
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     promoter        complement(5136..5240)
                     /label=AmpR promoter
     rep_origin      complement(5576..6031)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
       61 tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg ctagcatgac
      121 tggtggacag caaatgggtc ggatccggct gctaacaaag cccgaaagga agctgagttg
      181 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      241 aggggttttt tgctgaaagg aggaactata tccggatctg cattaatgaa tcggccaacg
      301 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
      361 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
      421 atccacagaa tcaggggata acgcaggaaa gaacatgaat taattctcat gtttgacagc
      481 ttatcatcga ttagctttaa tgcggtagtt tatcacagtt aaattgctaa cgcagtcagg
      541 caccgtgtat gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc accctggatg
      601 ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat atcgtccatt
      661 ccgacagcat cgccagtcac tatggcgtgc tgctagcgct atatgcgttg atgcaatttc
      721 tatgcgcacc cgttctcgga gcactgtccg accgctttgg ccgccgccca gtcctgctcg
      781 cttcgctact tggagccact atcgactacg cgatcatggc gaccacaccc gtcctgtgga
      841 ttctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
      901 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
      961 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     1021 ccttacatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     1081 gcttcctaat gcaggagtcg cataagggag agcgccgacc gatgcccttg agagccttca
     1141 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     1201 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg
     1261 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct
     1321 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc
     1381 aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga
     1441 cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc atcgggatgc
     1501 ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga cagcttcaag
     1561 gatcgctcgc ggctcttacc agcctaactt cgatcactgg accgctgatc gtcacggcga
     1621 tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc gccgccctat
     1681 accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa
     1741 tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt
     1801 cttgcggaga actgtgaatg cgcaaaccaa cccttggcaa ctgttgggaa gggcgatcgg
     1861 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     1921 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaatccg
     1981 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2041 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2101 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2161 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     2221 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     2281 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     2341 tgacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     2401 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     2461 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     2521 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     2581 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     2641 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     2701 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     2761 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     2821 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     2881 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     2941 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     3001 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     3061 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     3121 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     3181 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     3241 cataccgcga aaggttttgc accattcgat ggtgtcggaa tttatgcggt gtgaaatacc
     3301 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     3361 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3421 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3481 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3541 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3601 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3661 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3721 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3781 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3841 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3901 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3961 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4021 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4081 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4141 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4201 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4261 gtaaactttg gctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4321 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4381 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4441 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4501 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4561 agttaatagt ttgcgcaacg ttgttgccat tgcggcatcg tggtgtcacg ctcgtcgttt
     4621 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4681 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4741 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4801 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4861 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     4921 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4981 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5041 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5101 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5161 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5221 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     5281 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     5341 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     5401 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     5461 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     5521 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgaaatt
     5581 gtaaacgtta atattttgtt aaaattcgcg ttaaatattt gttaaatcag ctcatttttt
     5641 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5701 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5761 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc acccaaatca
     5821 agttttttgc ggtcgaggtg ccgtaaagct ctaaatcgga accctaaagg gagcccccga
     5881 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5941 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     6001 gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt
     6061 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca g
//
