LOCUS       Exported                6295 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pLoxGentrc, complete sequence.
ACCESSION   JF934954
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6295)
  AUTHORS   Palmeros B, Wild J, Szybalski W, Le Borgne S, Hernandez-Chavez G, 
            Gosset G, Valle F, Bolivar F.
  TITLE     A family of removable cassettes designed to obtain 
            antibiotic-resistance-free genomic modifications of Escherichia coli
            and other bacteria
  JOURNAL   Gene 247 (1-2), 255-264 (2000)
  PUBMED    10773465
REFERENCE   2  (bases 1 to 6295)
  AUTHORS   Sabido A, Martinez LM, de Anda R, Martinez A, Bolivar F, Gosset G.
  TITLE     A novel plasmid vector designed for chromosomal gene integration and
            expression: Use for developing a genetically stable Escherichia coli
            melanin production strain
  JOURNAL   Plasmid 69 (1), 16-23 (2013)
  PUBMED    22884755
REFERENCE   3  (bases 1 to 6295)
  AUTHORS   Sabido A, Gosset G.
  TITLE     Direct Submission
  JOURNAL   Submitted (11-MAY-2011) Cell Engineering and Biocatalysis, 
            Biotechnology Institute, Av. Universidad 2001, Cuernavaca, Morelos 
            62210, Mexico
REFERENCE   4  (bases 1 to 6295)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6295
                     /organism="Expression vector pLoxGentrc"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /note="derivative of vector pLoxGen4; numbered such that 
                     the first nucleotide is the last nucleotide of intergenic 
                     region of M13 bacteriophage and the count increases 
                     clockwise; carries ampicillin (bla) and gentamycin (aacC1) 
                     resistance genes; two parallel loxP sites from pSL1180 are 
                     flanking the aacC1 gene; contains all sequences for the 
                     correct expression of a gene from plasmid pTrc99A; carries 
                     a ColE1 and M13 bacteriophage replication origins; used for
                     the integration of genes into Escherichia coli"
                     /db_xref="taxon:1031331"
     misc_feature    1..474
                     /note="intergenic region of M13 bacteriophage; includes all
                     necessary elements for single strand replication in M1 
                     bacteriophage DNA"
     rep_origin      complement(1..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     669..685
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    688..762
                     /note="Multiple Cloning Site (MCS) modified from plasmid 
                     pLoxGen4; contains the following restriction sites: PmlI, 
                     SfiI, BalI, SpeI, ScaI, XhoI, SalI, ClaI and BamHI; HindIII
                     site was eliminated"
     primer_bind     733..749
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(763..796)
                     /note="loxP site; the 34 bp loxP sequence is the DNA target
                     site for Cre site-specific recombinase from P1 
                     bacteriophage; the two loxP sequences are flanking the 
                     resistance gene; therefore, antibiotic resistance gene can 
                     be eliminated by Cre trans-action"
     protein_bind    complement(763..796)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     gene            complement(840..1590)
                     /gene="aacC1"
                     /label=aacC1
     CDS             complement(840..1373)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /function="confers resistance to gentamycin"
                     /label=aacC1
                     /protein_id="AEG75266.1"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     regulatory      complement(1557..1563)
                     /regulatory_class="minus_10_signal"
                     /gene="aacC1"
     promoter        complement(1562..1590)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     regulatory      complement(1585..1590)
                     /regulatory_class="minus_35_signal"
                     /gene="aacC1"
     misc_feature    complement(1599..1632)
                     /note="loxP site; DNA target site for Cre site-specific 
                     recombinase"
     protein_bind    complement(1599..1632)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    complement(1697..2122)
                     /note="derived from the rrnB operon; the entire operon is 
                     not contained in this vector"
     gene            complement(1716..2041)
                     /gene="rrnB"
                     /label=rrnB
     regulatory      complement(1716..1743)
                     /regulatory_class="terminator"
                     /gene="rrnB"
                     /note="rrnB T2"
     terminator      1716..1743
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      1835..1921
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     regulatory      complement(1875..1918)
                     /regulatory_class="terminator"
                     /gene="rrnB"
                     /note="rrnB T1"
     rRNA            complement(1912..2041)
                     /gene="rrnB"
                     /product="5S ribosomal RNA"
                     /label=rrnB
     misc_feature    complement(2122..2186)
                     /note="Multiple Cloning Site (MCS) from plasmid pTrc99A; 
                     contains the following restriction sites: NcoI, EcoRI, 
                     SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, and HindIII"
     misc_feature    2124..2180
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     regulatory      complement(2187..2432)
                     /regulatory_class="promoter"
                     /note="trc promoter from plasmid pTrc99A; combines the -35 
                     region from the trp promoter and the -10 region from the 
                     lacUV5 promoter"
     regulatory      complement(2192..2195)
                     /regulatory_class="ribosome_binding_site"
                     /note="from lacZ"
     protein_bind    2204..2220
                     /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(2228..2257)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     regulatory      complement(2229..2234)
                     /regulatory_class="minus_10_signal"
                     /note="from trp promoter"
     regulatory      complement(2252..2257)
                     /regulatory_class="minus_35_signal"
                     /note="from lacUV5 promoter"
     gene            complement(2463..3649)
                     /gene="lacI"
                     /allele="q"
                     /label=lacI
                     /note="lacIq allele has been added to render the vector 
                     autorepressed and to expand to their host range to 
                     non-lacIq strains"
     CDS             complement(2489..3571)
                     /codon_start=1
                     /gene="lacI"
                     /allele="q"
                     /product="LacI repressor"
                     /label=lacI
                     /note="transcriptional repressor of the lac operon"
                     /protein_id="AEG75265.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(3572..3649)
                     /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."
     regulatory      complement(3607..3612)
                     /regulatory_class="minus_10_signal"
                     /gene="lacI"
                     /allele="q"
     regulatory      complement(3630..3635)
                     /regulatory_class="minus_35_signal"
                     /gene="lacI"
                     /allele="q"
     misc_feature    complement(3650..3680)
                     /label=lacIq 5 sequence
                     /note="lacIq 5 sequence"
     misc_feature    complement(3681..3873)
                     /label=intergenic region from pTrc99A
                     /note="intergenic region from pTrc99A"
     primer_bind     complement(3888..3904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3912..3928
                     /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(3936..3966)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     misc_feature    3980..4920
                     /note="replication region from pLoxGen4 plasmid; all 
                     elements needed for double strand DNA replication are 
                     included"
     protein_bind    3981..4002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      4284..4292
     rep_origin      complement(4290..4878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     gene            complement(5049..5979)
                     /gene="bla"
                     /label=bla
     CDS             complement(5049..5909)
                     /codon_start=1
                     /gene="bla"
                     /product="penicillin beta-lactamase"
                     /function="confers resistance to ampicillin"
                     /label=bla
                     /protein_id="AEG75267.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5910..6014)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      complement(5951..5956)
                     /regulatory_class="minus_10_signal"
                     /gene="bla"
     regulatory      complement(5974..5979)
                     /regulatory_class="minus_35_signal"
                     /gene="bla"