LOCUS       40924_28571        3443 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Integration vector pLOI2227, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3443)
  AUTHORS   Martinez-Morales F, Borges AC, Martinez A, Shanmugam KT, Ingram LO.
  TITLE     Chromosomal integration of heterologous DNA in Escherichia coli with
            precise removal of markers and replicons used during construction
  JOURNAL   J. Bacteriol. 181 (22), 7143-7148 (1999)
  PUBMED    10559184
REFERENCE   2  (bases 1 to 3443)
  AUTHORS   Borges AC, Martinez-Morales F, Ingram LO, Shanmugam KT.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-JUL-1999) Microbiology and Cell Science, University of
            Florida, 981 Museum Road, Room 1160, Gainesville, FL 32611-0700, USA
REFERENCE   3  (bases 1 to 3443)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3443)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. 
            Bacteriol."; date: "1999"; volume: "181"; issue: "22"; pages: 
            "7143-7148"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (28-JUL-1999) Microbiology and Cell Science, University of Florida, 
            981 Museum Road, Room 1160, Gainesville, FL 32611-0700, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..50
                     /label=FRT (minimal)
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     promoter        86..104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    112..182
                     /label=multiple cloning site
                     /note="multiple cloning site"
     protein_bind    214..248
                     /label=flipase binding site
                     /bound_moiety="flipase"
                     /note="flipase-binding site"
     protein_bind    216..249
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     CDS             complement(415..1362)
                     /codon_start=1
                     /label=Rep101(Ts)
                     /note="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      complement(1410..1632)
                     /direction=LEFT
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(2501..3292)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
ORIGIN
        1 atcgatgaat tgatccgaag ttcctattct ctagaaagta taggaacttc gaattgtcga
       61 caagcttgat ctggcttatc gaaattaata cgactcacta tagggagacc ggaattccga
      121 gcttggcgcg ccaggtcgac tctagaggat ccccggggaa gatcttccgg aagatcttcc
      181 cgagctcgaa ttaattccgc gatgaattga tcccggaagt tcctattctc tagaaagtat
      241 aggaacttcg aattggtcga caagctagct tgcatgcaag cttggcactg gctgatcagc
      301 tagcccatgg gtatggacag ttttcccttt gatatgtaac ggtgaacagt tgttctactt
      361 ttgtttgtta gtcttgatgc ttcactgata gatacaagag ccataagaac ctcagatcct
      421 tccgtattta gccagtatgt tctctagtgt ggttcgttgt ttttgcgtga gccatgagaa
      481 cgaaccattg agatcatgct tactttgcat gtcactcaaa aattttgcct caaaactggt
      541 gagctgaatt tttgcagtta aagcatcgtg tagtgttttt cttagtccgt tacgtaggta
      601 ggaatctgat gtaatggttg ttggtatttt gtcaccattc atttttatct ggttgttctc
      661 aagttcggtt acgagatcca tttgtctatc tagttcaact tggaaaatca acgtatcagt
      721 cgggcggcct cgcttatcaa ccaccaattt catattgctg taagtgttta aatctttact
      781 tattggtttc aaaacccatt ggttaagcct tttaaactca tggtagttat tttcaagcat
      841 taacatgaac ttaaattcat caaggctaat ctctatattt gccttgtgag ttttcttttg
      901 tgttagttct tttaataacc actcataaat cctcatagag tatttgtttt caaaagactt
      961 aacatgttcc agattatatt ttatgaattt ttttaactgg aaaagataag gcaatatctc
     1021 ttcactaaaa actaattcta atttttcgct tgagaacttg gcatagtttg tccactggaa
     1081 aatctcaaag cctttaacca aaggattcct gatttccaca gttctcgtca tcagctctct
     1141 ggttgcttta gctaatacac cataagcatt ttccctactg atgttcatca tctgaacgta
     1201 ttggttataa gtgaacgata ccgtccgttc tttccttgta gggttttcaa tcgtggggtt
     1261 gagtagtgcc acacagcata aaattagctt ggtttcatgc tccgttaagt catagcgact
     1321 aatcgctagt tcatttgctt tgaaaacaac taattcagac atacatctca attggtctag
     1381 gtgattttaa tcactatacc aattgagatg ggctagtcaa tgataattac tagtcctttt
     1441 cctttgagtt gtgggtatct gtaaattctg ctagaccttt gctggaaaac ttgtaaattc
     1501 tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt ttttttgttt atattcaagt
     1561 ggttataatt tatagaataa agaaagaata aaaaaagata aaaagaatag atcccagccc
     1621 tgtgtataac tcactacttt agtcagttcc gcagtattac aaaaggatgt cgcaaacgct
     1681 gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc ttaagtagca ccctcgcaag
     1741 ctcggttgcg gccgcaatcg ggcaaatcgc tgaatattcc ttttgtctcc gaccatcagg
     1801 cacctgagtc gctgtctttt tcgtgacatt cagttcgctg cgctcacggc tctggcagtg
     1861 aatgggggta aatggcacta caggcgcctt ttatggattc atgcaaggaa actacccata
     1921 atacaagaaa agcccgtcac gggcttctca gggcgtttta tggcgggtct gctatgtggt
     1981 gctatctgac tttttgctgt tcagcagttc ctgccctctg attttccagt ctgaccactt
     2041 cggattatcc cgtgacaggt cattcagact ggctaatgca cccagtaagg cagcggtatc
     2101 atcaacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2161 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2221 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2281 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2341 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2401 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccgggccgc aaaaattaaa
     2461 aatgaagttt tgacggtatc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc
     2521 gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt
     2581 cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat
     2641 agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca
     2701 ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca
     2761 tccgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
     2821 gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt
     2881 tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat
     2941 cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg
     3001 gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
     3061 cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct tggagttcat
     3121 tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc
     3181 ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc
     3241 tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg
     3301 atcctcatcc tgtctcttga tccactagat tattgaagca tttatcaggg ttattgtctc
     3361 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3421 tttccccgaa aagtgccacc tgc
//
