LOCUS       40924_28606        6295 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pLoxGentrc, complete sequence.
ACCESSION   .
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)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6295)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Gene 247 
            (1-2), 255-264 (2000)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Plasmid"; 
            date: "2013"; volume: "69"; issue: "1"; pages: "16-23"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (11-MAY-2011) Cell Engineering and Biocatalysis, Biotechnology 
            Institute, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     primer_bind     733..749
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    763..796
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(843..1373)
                     /label=GmR
                     /note="gentamycin acetyltransferase"
     regulatory      complement(1557..1563)
                     /gene="aacC1"
                     /regulatory_class="minus_10_signal"
     promoter        complement(1562..1590)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     regulatory      complement(1585..1590)
                     /gene="aacC1"
                     /regulatory_class="minus_35_signal"
     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)."
     terminator      complement(1716..1743)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(1835..1921)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     rRNA            complement(1912..2041)
                     /gene="rrnB"
                     /product="5S ribosomal RNA"
                     /label=rrnB
     misc_feature    complement(2124..2180)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     regulatory      complement(2192..2195)
                     /label=from lacZ
                     /note="from lacZ"
                     /regulatory_class="ribosome_binding_site"
     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"
     CDS             complement(2492..3571)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(3572..3649)
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     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    complement(3912..3928)
                     /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(3936..3966)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3981..4002)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4290..4878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5052..5909)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5910..6014)
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg
      481 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
      541 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
      601 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
      661 acgacgttgt aaaacgacgg ccagtgccaa gctagcttaa ggtgcacggc ccacgtggcc
      721 actagtactt ctcgaggtcg acggtatcga taagctggat ccataacttc gtataatgta
      781 tgctatacga agttatctag cgcgtcggcc gggaagccga tctcggcttg aacgaattgt
      841 taggtggcgg tacttgggtc gatatcaaag tgcatcactt cttcccgtat gcccaacttt
      901 gtatagagag ccactgcggg atcgtcaccg taatctgctt gcacgtagat cacataagca
      961 ccaagcgcgt tggcctcatg cttgaggaga ttgatgagcg cggtggcaat gccctgcctc
     1021 cggtgctcgc cggagactgc gagatcatag atatagatct cactacgcgg ctgctcaaac
     1081 ctgggcagaa cgtaagccgc gagagcgcca acaaccgctt cttggtcgaa ggcagcaagc
     1141 gcgatgaatg tcttactacg gagcaagttc ccgaggtaat cggagtccgg ctgatgttgg
     1201 gagtaggtgg ctacgtctcc gaactcacga ccgaaaagat caagagcagc ccgcatggat
     1261 ttgacttggt cagggccgag cctacatgtg cgaatgatgc ccatacttga gccacctaac
     1321 tttgttttag ggcgactgcc ctgctgcgta acatcgttgc tgctgcgtaa catcgttgct
     1381 gctccataac atcaaacatc gacccacggc gtaacgcgct tgctgcttgg atgcccgagg
     1441 catagactgt acaaaaaaac agtcataaca agccatgaaa accgccactg cgccgttacc
     1501 accgctgcgt tcggtcaagg ttctggacca gttgcgtgag cgcatacgct acttgcatta
     1561 cagtttacga accgaacagg cttatgtcga cgcgctagat aacttcgtat aatgtatgct
     1621 atacgaagtt atgcggccgc gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     1681 tagaaaaata aacaaaaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct
     1741 tctgcttaat ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc
     1801 gttgcttcgc aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca
     1861 ccgacaaaca acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt
     1921 gatgcctggc agttccctac tctcgcatgg ggagacccca cactaccatc ggcgctacgg
     1981 cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc
     2041 aaattctgtt ttatcagacc gcttctgcgt tctgatttaa tctgtatcag gctgaaaatc
     2101 ttctctcatc cgccaaaaca gccaagcttg catgcctgca ggtcgactct agaggatccc
     2161 cgggtaccga gctcgaattc catggtctgt ttcctgtgtg aaattgttat ccgctcacaa
     2221 ttccacacat tatacgagcc ggatgattaa ttgtcaacag ctcatttcag aatatttgcc
     2281 agaaccgtta tgatgtcggc gcaaaaaaca ttatccagaa cgggagtgcg ccttgagcga
     2341 cacgaattat gcagtgattt acgacctgca cagccatacc acagcttccg atggctgcct
     2401 gacgccagaa gcattggtgc accgtgcagt cgatgataag ctgtcaaacc agatcaattc
     2461 gcgctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2521 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2581 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     2641 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     2701 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     2761 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     2821 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     2881 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     2941 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     3001 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     3061 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     3121 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     3181 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     3241 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     3301 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     3361 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     3421 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     3481 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     3541 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     3601 ggcgctatca tgccataccg cgaaaggttt tgcaccattc gatggtgtca acgtaaatgc
     3661 atgccgcttc gccttcgcgc gcgaattgat ctgctgcctc gcgcgtttcg gtgatgacgg
     3721 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     3781 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     3841 catgacccag tcacgtagcg atagcggagt gtaaattcgt aatcatggtc atagctgttt
     3901 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3961 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4021 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4081 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4141 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4201 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4261 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4321 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4381 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4441 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4501 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4561 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4621 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4681 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4741 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4801 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4861 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4921 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4981 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5041 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5101 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5161 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5221 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5281 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5341 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5401 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5461 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5521 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5581 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5641 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5701 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5761 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5821 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5881 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5941 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6001 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     6061 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     6121 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     6181 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     6241 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccata
//
