LOCUS       sequence-418610-        5905 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 5905)
  TITLE     2-series LIC N-terminal fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 5905)
  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 (2M-T)
FEATURES             Location/Qualifiers
     source          1..5905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(57..62,63..79,80..85))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(112..130)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        198..302
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(303..371,372..1163)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(521..540)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1334..1922
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1823..1842
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2076..2093
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2108..2247
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2173..2192)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2333..2355
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2349..2540)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3998..4017
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4101..4120
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4101..4119
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4208..4230
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4250..4267
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4277..5377
                     /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     5351..5374
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5390..5410
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5535..5553)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5549..5596
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta
       61 aagcttatcg atgataagct gtcaaacatg agaattcttg aagacgaaag ggcctcgtga
      121 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
      181 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      241 tgtatccgct catgagacaa taaccctgat aaatgcttca ataacattga aaaaggaaga
      301 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
      361 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
      421 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
      481 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
      541 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
      601 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
      661 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
      721 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
      781 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
      841 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
      901 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
      961 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     1021 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     1081 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     1141 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     1201 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     1261 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1321 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1381 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1441 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     1501 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     1561 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     1621 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     1681 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     1741 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     1801 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     1861 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     1921 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     1981 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     2041 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     2101 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     2161 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     2221 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     2281 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     2341 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     2401 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     2461 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     2521 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     2581 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     2641 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     2701 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     2761 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     2821 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     2881 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     2941 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     3001 acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct ggagatggcg
     3061 gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat
     3121 tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg gcttccattc
     3181 aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata
     3241 gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg
     3301 ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt
     3361 gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca
     3421 tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg
     3481 cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct
     3541 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     3601 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     3661 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     3721 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     3781 aggctctcaa gggcatcggt cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     3841 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     3901 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     3961 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     4021 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     4081 agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc
     4141 tagtgccggc tccggagagc tctttaatta agcggccgcc ctgcaggact cgagttctag
     4201 aaataatttt gtttaacttt aagaaggaga tatacatatg aaatcttctc accatcacca
     4261 tcaccatggt tcttctatga aaatcgaaga aggtaaactg gtaatctgga ttaacggcga
     4321 taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata ccggaattaa
     4381 agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg cggcaactgg
     4441 cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg
     4501 cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc cgtttacctg
     4561 ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg aagcgttatc
     4621 gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag agatcccggc
     4681 gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc
     4741 gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt atgaaaacgg
     4801 caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt
     4861 cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact ccatcgcaga
     4921 agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg catggtccaa
     4981 catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca agggtcaacc
     5041 atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc cgaacaaaga
     5101 gctggcaaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg aagcggttaa
     5161 taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga
     5221 tccacgtatt gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc
     5281 gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg
     5341 tcagactgtc gatgaagccc tgaaagacgc gcagactaat gggatcgagg aaaacctgta
     5401 cttccaatcc aatattggaa gtggataacg gatccgcgat cgcggcgcgc cacctggtgg
     5461 ccggccggta ccacgcgtgc gcgctgatcc ggctgctaac aaagcccgaa aggaagctga
     5521 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     5581 cttgaggggt tttttgctga aaggaggaac tatatccgga tatccacagg acgggtgtgg
     5641 tcgccatgat cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc
     5701 ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat
     5761 atagcgctag cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca
     5821 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
     5881 cgaggatgac gatgagcgca ttgtt
//
