LOCUS       sequence-174752-        7270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET LIC cloning vector for Biotin-His6-MBP-N10-tev-yORF-mClover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7270)
  TITLE     C-terminal fluorescent fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 7270)
  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 mClover LIC cloning vector (MBP-mClover)
FEATURES             Location/Qualifiers
     source          1..7270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     91..110
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     194..213
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        194..212
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    213..237
                     /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             252..274
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             296..340
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             356..373
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             383..1483
                     /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     1457..1480
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1538..1558
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1595..1597,1598..2308)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1637..1658)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2245..2266
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2327..2344
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2397..2415)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2411..2458
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2495..2950
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2582..2601)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2792..2813
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3043..3858)
                     /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(3043..3858)
                     /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     3766..3785
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3850..3869
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3980..4568
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4469..4488
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4722..4739
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4754..4893
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4819..4838)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4979..5001
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4995..5186)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5995..7077)
                     /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"
     primer_bind     7039..7058
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7078..7155)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
       61 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
      121 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
      181 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
      241 ctagaaataa ttttgtttaa ctttaagaag gagatatagc atgggttctt ctggaggcct
      301 gaacgatatt tttgaagcgc agaaaattga atggcatgaa tccatgggtt cttctcacca
      361 tcaccatcac cacggttctt ctatgaaaat cgaagaaggt aaactggtaa tctggattaa
      421 cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg
      481 aattaaagtc accgttgagc atccggataa actggaagag aaattcccac aggttgcggc
      541 aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca
      601 atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt
      661 tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc
      721 gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat
      781 cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca
      841 agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga
      901 aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct
      961 gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat
     1021 cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg
     1081 gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg
     1141 tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa
     1201 caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc
     1261 ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggc
     1321 gaaagatcca cgtattgccg ccactatgga aaacgcccag aaaggtgaaa tcatgccgaa
     1381 catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag
     1441 cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga gctcgaacaa
     1501 caacaacaat aacaataaca acaacctcgg gatcgaggaa aacctgtact tccaatccaa
     1561 tattggtagt gggagcaacg gcagcagcgg atccgtgagc aagggcgagg agctgttcac
     1621 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1681 ccgcggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac
     1741 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcgtggc
     1801 ctgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1861 cgaaggctac gtccaggagc gcaccatctc tttcaaggac gacggtacct acaagacccg
     1921 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1981 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca acagccacaa
     2041 cgtctatatc acggccgaca agcagaagaa cggcatcaag gctaacttca agatccgcca
     2101 caacgttgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2161 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc catcagtccg ccctgagcaa
     2221 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2281 tacacatggc atggacgagc tgtacaagta agcggccgca ctcgagcacc accaccacca
     2341 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     2401 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2461 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2521 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2581 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2641 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2701 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2761 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2821 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2881 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa actagtaacg
     2941 tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3001 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     3061 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     3121 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     3181 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     3241 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     3301 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     3361 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     3421 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     3481 cactgccagc gcatcaacaa tgttttcacc tgaatcagga tattcttcta atacctggaa
     3541 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     3601 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     3661 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     3721 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     3781 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     3841 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     3901 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3961 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4021 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4081 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4141 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4201 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4261 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4321 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4381 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4441 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4501 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4561 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4621 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4681 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4741 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4801 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4861 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4921 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4981 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     5041 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     5101 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     5161 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     5221 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     5281 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     5341 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     5401 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     5461 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     5521 gtcgcagacg ttttgcagaa gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     5581 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     5641 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     5701 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     5761 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     5821 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     5881 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5941 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     6001 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6061 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     6121 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     6181 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     6241 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     6301 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     6361 gggaacgata ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     6421 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     6481 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     6541 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     6601 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     6661 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     6721 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     6781 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     6841 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     6901 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     6961 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     7021 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     7081 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     7141 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     7201 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     7261 agatggcgcc
//
