LOCUS       sequence-237947-        6462 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6462)
  TITLE     LIC N-terminal fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 6462)
  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 His6 MBP TEV LIC cloning vector (1M)
FEATURES             Location/Qualifiers
     source          1..6462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             335..1435
                     /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     1409..1432
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1448..1468
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1534..1551
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1604..1622)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1618..1665
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1702..2157
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1789..1808)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1999..2020
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2250..3065)
                     /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(2250..3065)
                     /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     2973..2992
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3057..3076
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3187..3775
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3676..3695
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3929..3946
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3961..4100
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4026..4045)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4186..4208
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4202..4393)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5202..6284)
                     /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     6246..6265
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6285..6362)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 ttcttctcac catcaccatc accatggttc ttctatgaaa atcgaagaag gtaaactggt
      361 aatctggatt aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga
      421 gaaagatacc ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc
      481 acaggttgcg gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg
      541 tggctacgct caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa
      601 gctgtatccg tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat
      661 cgctgttgaa gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac
      721 ctgggaagag atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat
      781 gttcaacctg caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc
      841 gttcaagtat gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc
      901 gaaagcgggt ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac
      961 cgattactcc atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg
     1021 cccgtgggca tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc
     1081 gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc
     1141 cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga
     1201 aggtctggaa gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga
     1261 ggaagagttg gcgaaagatc cacgtattgc cgccactatg gaaaacgccc agaaaggtga
     1321 aatcatgccg aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat
     1381 caacgccgcc agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaatgg
     1441 gatcgaggaa aacctgtact tccaatccaa tattggaagt ggataacgga tccgaattcg
     1501 agcgccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     1561 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1621 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1681 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1741 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1801 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1861 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1921 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1981 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2041 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2101 agctgattta acaaaaattt aacgcgaatt ttaacaaact agtaacgttt acaatttcag
     2161 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2221 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     2281 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     2341 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     2401 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     2461 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     2521 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     2581 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     2641 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     2701 tcaacaatgt tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     2761 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     2821 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     2881 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     2941 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3001 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     3061 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     3121 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3181 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3241 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3301 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3361 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3421 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3481 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3541 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3601 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3661 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3721 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3781 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3841 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3901 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3961 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     4021 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4081 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4141 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4201 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     4261 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     4321 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     4381 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     4441 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     4501 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4561 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4621 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4681 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     4741 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     4801 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     4861 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     4921 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     4981 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5041 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5101 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5161 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5221 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5281 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     5341 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     5401 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     5461 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     5521 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgataccc
     5581 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     5641 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     5701 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     5761 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     5821 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     5881 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     5941 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6001 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6061 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6121 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6181 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6241 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     6301 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     6361 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     6421 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tg
//
