LOCUS       sequence-174748-        7264 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET LIC cloning vector for Biotin-His6-MBP-N10-tev-yORF-mRuby2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7264)
  TITLE     C-terminal fluorescent fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 7264)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pET MBP mRuby2 LIC cloning vector (MBP-mRuby2)
FEATURES             Location/Qualifiers
     source          1..7264
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     618..637
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(657..734)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     940..959
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1043..1062
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1043..1061
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1062..1086
                     /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)."
     RBS             1101..1123
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1145..1189
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1205..1222
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1232..2332
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2306..2329
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2387..2407
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2444..3151
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     CDS             3170..3187
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3240..3258)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3254..3301
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3338..3793
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3425..3444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3635..3656
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3886..4701)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3886..4701)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4609..4628
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4693..4712
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4823..5411
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5312..5331
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5565..5582
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5597..5736
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5662..5681)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5822..5844
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5838..6029)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6838..7264,1..656))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
       61 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
      121 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
      181 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
      241 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
      301 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
      361 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
      421 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
      481 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
      541 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
      601 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
      661 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
      721 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
      781 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
      841 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
      901 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
      961 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     1021 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     1081 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatagca tgggttcttc
     1141 tggaggcctg aacgatattt ttgaagcgca gaaaattgaa tggcatgaat ccatgggttc
     1201 ttctcaccat caccatcacc acggttcttc tatgaaaatc gaagaaggta aactggtaat
     1261 ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa
     1321 agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca
     1381 ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
     1441 ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct
     1501 gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc
     1561 tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
     1621 ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt
     1681 caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt
     1741 caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
     1801 agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga
     1861 ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
     1921 gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac
     1981 cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc
     2041 cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg
     2101 tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
     2161 agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga aaggtgaaat
     2221 catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa
     2281 cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag
     2341 ctcgaacaac aacaacaata acaataacaa caacctcggg atcgaggaaa acctgtactt
     2401 ccaatccaat attggtagtg ggagcaacgg cagcagcgga tccgtgtcta agggcgaaga
     2461 gctgatcaag gaaaatatgc gtatgaaggt ggtcatggaa ggttcggtca acggccacca
     2521 attcaaatgc acaggtgaag gagaaggcaa tccgtacatg ggaactcaaa ccatgaggat
     2581 caaagtcatc gagggaggac ccctgccatt tgcctttgac attcttgcca cgtcgttcat
     2641 gtatggcagc cgtactttta tcaagtaccc gaaaggcatt cctgatttct ttaaacagtc
     2701 ctttcctgag ggttttactt gggaaagagt tacgagatac gaagatggtg gagtcgtcac
     2761 cgtcatgcag gacaccagcc ttgaggatgg ctgtctcgtt taccacgtcc aagtcagagg
     2821 ggtaaacttt ccctccaatg gtcccgtgat gcagaagaag accaagggtt gggagcctaa
     2881 tacagagatg atgtatccag cagatggtgg tctgagggga tacactcata tggcactgaa
     2941 agttgatggt ggtggccatc tgtcttgctc tttcgtaaca acttacaggt caaaaaagac
     3001 cgtcgggaac atcaagatgc ccggtatcca tgccgttgat caccgcctgg aaaggttaga
     3061 ggaaagtgac aatgaaatgt tcgtagtaca acgcgaacac gcagttgcca agttcgccgg
     3121 gcttggtggt gggatggacg agctgtacaa gtaagcggcc gcactcgagc accaccacca
     3181 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     3241 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     3301 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     3361 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3421 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3481 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3541 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3601 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3661 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3721 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaactagta
     3781 acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3841 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     3901 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     3961 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     4021 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     4081 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     4141 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     4201 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     4261 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     4321 gaacactgcc agcgcatcaa caatgttttc acctgaatca ggatattctt ctaatacctg
     4381 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     4441 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     4501 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     4561 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     4621 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     4681 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     4741 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4801 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4861 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4921 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4981 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5041 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5101 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5161 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5221 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5281 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5341 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5401 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5461 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     5521 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     5581 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5641 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     5701 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     5761 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5821 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5881 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     5941 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     6001 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     6061 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     6121 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     6181 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     6241 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     6301 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     6361 caggtcgcag acgttttgca gaagcagtcg cttcacgttc gctcgcgtat cggtgattca
     6421 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     6481 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     6541 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     6601 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     6661 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     6721 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     6781 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     6841 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6901 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     6961 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     7021 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     7081 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     7141 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     7201 agtgggaacg ataccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     7261 ccag
//
