LOCUS       40924_28761        3792 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pLSP2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3792)
  AUTHORS   Lehman SS, Mladinich KM, Boonyakanog A, Mima T, Karkhoff-Schweizer 
            RR, Schweizer HP.
  TITLE     Versatile nourseothricin and streptomycin/spectinomycin resistance 
            gene cassettes and their use in chromosome integration vectors
  JOURNAL   J. Microbiol. Methods 129, 8-13 (2016)
  PUBMED    27457407
REFERENCE   2  (bases 1 to 3792)
  AUTHORS   Lehman SS, Mladinich K, Boonyakanog A, Mima T, Karkhoff-Schweizer 
            RR, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-MAR-2016) Molecular Genetics and Microbiology, 
            University of Florida, 2055 Mowry Road, Gainesville, FL 32610, USA
REFERENCE   3  (bases 1 to 3792)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3792)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. 
            Microbiol. Methods 129, 8-13 (2016)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (17-MAR-2016) Molecular Genetics and Microbiology, University of 
            Florida, 2055 Mowry Road, Gainesville, FL 32610, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..3792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(184..772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(946..1803)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(1804..1908)
                     /label=AmpR promoter
     primer_bind     2380..2396
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2400..2444
                     /note="MCS; multiple cloning site"
     misc_feature    2450..2482
                     /note="loxP; Cre recombinase site"
     regulatory      2550..2603
                     /label=chloramphenicol transacetylase gene promoter
                     /note="chloramphenicol transacetylase gene promoter"
                     /regulatory_class="promoter"
     regulatory      2567..2572
                     /regulatory_class="minus_35_signal"
     regulatory      2590..2595
                     /regulatory_class="minus_10_signal"
     regulatory      2637..2641
                     /regulatory_class="ribosome_binding_site"
     CDS             2649..3401
                     /codon_start=1
                     /gene="aad9"
                     /product="spectinomycin adenyltransferase"
                     /label=aad9
                     /protein_id="ANY60775.1"
                     /translation="MNTYEQINKVKKILRKHLKNNLIGTYMFGSGVESGLKPNSDLDFL
                     VVVSEPLTDQSKEILIQKIRPISKKIGDKSNLRYIELTIIIQQEMVPWNHPPKQEFIYG
                     EWLQELYEQGYIPQKELNSDLTIMLYQAKRKNKRIYGNYDLEELLPDIPFSDVRRAIMD
                     SSEELIDNYQDDETNSILTLCRMILTMDTGKIIPKDIAGNAVAESSPLEHRERILLAVR
                     SYLGENIEWTNENVNLTINYLNNRLKKL"
     gene            2649..3401
                     /gene="aad9"
                     /label=aad9
                     /note="codon optimized; derived from Enterococcus faecalis"
     regulatory      3402..3443
                     /regulatory_class="terminator"
     protein_bind    complement(3479..3512)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3517..3561
                     /note="MCS; multiple cloning site"
     primer_bind     complement(3574..3590)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3598..3614)
                     /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(3622..3652)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3667..3688)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
ORIGIN
        1 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
       61 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      601 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
      661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1921 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     1981 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     2041 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2101 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     2161 agagcagatt gtactgagag tgcaccatgc ggtgtgaaat accgcacaga tgcgtaagga
     2221 gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     2281 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     2341 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca
     2401 agcttgcatg cctgcaggtc gactctagag gatccccggg tacccgcgtc tacttcgtat
     2461 agcatacatt atacgaagtt atactaaaac aattcatcca gtaaaatata atattttatt
     2521 ttctcccaat caggcttgat cccgaattca tgagacgttg atcggcacgt aagaggttcc
     2581 aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt atcctgagga
     2641 ggatttatat gaatacctat gaacagatta ataaagtgaa aaaaattctg cgcaaacatc
     2701 tgaaaaataa tctgattggc acctatatgt ttggcagcgg cgtggaaagc ggcctgaaac
     2761 cgaatagcga tctggatttt ctggtggtgg tgagcgaacc gctgaccgat cagagcaaag
     2821 aaattctgat tcagaaaatt cgcccgatta gcaaaaaaat tggcgataaa agcaatctgc
     2881 gctatattga actgaccatt attattcagc aggaaatggt gccgtggaat catccgccga
     2941 aacaggaatt tatttatggc gaatggctgc aagaactgta tgaacagggc tatattccgc
     3001 agaaagaact gaatagcgat ctgaccatta tgctgtatca ggccaaacgc aaaaataaac
     3061 gcatttatgg caattatgat ctggaagaac tgctgccgga tattccgttt agcgatgtgc
     3121 gccgcgccat tatggatagc agcgaagaac tgattgataa ttatcaggat gatgaaacca
     3181 atagcattct gaccctgtgc cgcatgattc tgaccatgga taccggcaaa attattccga
     3241 aagatattgc cggcaatgcc gtggccgaaa gcagcccgct ggaacaccgc gaacgcattc
     3301 tgctggccgt gcgcagctat ctgggcgaaa atattgaatg gaccaatgaa aatgtgaatc
     3361 tgaccattaa ttatctgaat aatcgcctga aaaaactgta aaaaaattga aaaaatggtg
     3421 gaaacacttt tttcaatttt tttgttttat tatgaattcc gacggagccg attttgaaat
     3481 aacttcgtat agcatacatt atacgaagtt atacgcggta cccggggatc ctctagagtc
     3541 gacctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3601 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     3661 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3721 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3781 gcgtattggg cg
//
