LOCUS       sequence-182500-        5959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  His6-MBP-N10-tev-yORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5959)
  TITLE     Series-371: N- or C-terminal Tag E.coli T7 Expression Vectors with 
            Biobrick Polycistronic Restriction Sites and Csy4 cleavage
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5959)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: 371-C
FEATURES             Location/Qualifiers
     source          1..5959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2080..2099)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2240..2262
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2256..2447)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3905..3924
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4008..4027
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4008..4026
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4092..4111
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             4136..4158
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4178..4195
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4205..5305
                     /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     5279..5302
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5360..5380
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5495..5513)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5509..5556
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5922..5927,5928..5944,5945..5950))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga ggcgcgccat gcggtatttt ctccttacgc
     2041 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     2101 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     2161 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2221 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2281 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     2341 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     2401 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     2461 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     2521 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     2581 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     2641 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     2701 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     2761 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     2821 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     2881 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
     2941 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
     3001 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
     3061 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     3121 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
     3181 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
     3241 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
     3301 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     3361 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     3421 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     3481 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     3541 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     3601 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     3661 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga cgctctccct
     3721 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     3781 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     3841 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     3901 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     3961 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     4021 tatagggaga ccacaacggt ttccctctag tgccggctcc ggagagctct ttaatgcggc
     4081 cgcttaatta agttcactgc cgtataggca gctaagaaaa ttaactagaa ataattttgt
     4141 ttaactttaa gaaggagata tacatatgaa atcttctcac catcaccatc accatggttc
     4201 ttctatgaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
     4261 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
     4321 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
     4381 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
     4441 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
     4501 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
     4561 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
     4621 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
     4681 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
     4741 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
     4801 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
     4861 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
     4921 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
     4981 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcaaaaga
     5041 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     5101 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
     5161 cgccactatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     5221 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
     5281 tgaagccctg aaagacgcgc agactaattc gagctcgaac aacaacaaca ataacaataa
     5341 caacaacctc gggatcgagg aaaacctgta cttccaatcc aatattggaa gtggataacg
     5401 gatcgcgatc gccctgcagg ccggccggta ccacgcgtgc gcgctgatcc ggctgctaac
     5461 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     5521 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     5581 tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg atagtggctc caagtagcga
     5641 agcgagcagg actgggcggc ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca
     5701 tagaaattgc atcaacgcat atagcgctag cagcacgcca tagtgactgg cgatgctgtc
     5761 ggaatggacg atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac
     5821 agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg
     5881 tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat
     5941 aagctgtcaa acatgagaa
//
