LOCUS       sequence-455388-        6905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of 10xHis-SUMO (Smt3) fused Avi-tag, HRV-3C 
            cleavable MSP2N2 for biotinylated nanodiscs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6905)
  TITLE     Molecular Toolbox
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6905)
  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            "Unpublished"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: MZB206
FEATURES             Location/Qualifiers
     source          1..6905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(781..1863)
                     /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     1825..1844
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1864..1941)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2147..2166
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2250..2269
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2250..2268
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2269..2293
                     /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             2308..2330
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2344..2373
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2674..2718
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2722..2745
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3886..3903
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3956..3974)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3970..4017
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4054..4509
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4141..4160)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4351..4372
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4535..4639
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4640..4708,4709..5500)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4858..4877)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5671..6259
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6160..6179
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6413..6430
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6445..6584
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6510..6529)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6670..6692
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6686..6877)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
       61 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
      121 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
      181 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
      241 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
      301 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
      361 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
      421 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
      481 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
      541 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
      601 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
      661 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
      721 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
      781 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
      841 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
      901 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
      961 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     1021 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     1081 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     1141 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     1201 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     1261 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     1321 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     1381 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     1441 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     1501 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     1561 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     1621 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     1681 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     1741 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     1801 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     1861 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     1921 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     1981 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     2041 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     2101 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     2161 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     2221 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     2281 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
     2341 ggccatcacc atcaccatca ccatcaccat cacggcagca tgagcgatag cgaagtgaac
     2401 caggaagcga aaccggaagt gaaaccggaa gtgaaaccgg aaacccatat taacctgaaa
     2461 gtgagcgatg gcagcagcga aatttttttt aaaattaaaa aaaccacccc gctgcgtcgt
     2521 ctgatggaag cgtttgcgaa acgtcagggc aaagaaatgg atagcctgcg ttttctgtat
     2581 gatggcattc gtattcaggc ggatcagacc ccggaagatc tggatatgga agataacgat
     2641 attattgaag cgcatcgtga acagattggc ggcggcctga acgatatttt tgaagcgcag
     2701 aaaattgaat ggcatgaagg tctggaagtg ctgtttcagg gtccgggtag cggttctacc
     2761 ttcagtaaac ttcgcgaaca actgggcccc gtgacgcagg aattctggga caacctggaa
     2821 aaagaaaccg agggactgcg tcaggaaatg tccaaagatt tagaagaggt gaaggccaag
     2881 gttcagccat atctcgatga ctttcagaaa aaatggcagg aagagatgga attatatcgt
     2941 caaaaggtgg aaccgctgcg tgcggaactg caagaggggg cacgccaaaa actccatgag
     3001 ctccaagaga agctcagccc attaggcgaa gaaatgcgcg atcgcgcccg tgcacatgtt
     3061 gatgcactcc ggactcattt ggcgccgtat tcggatgaac ttcgccagcg tttggccgca
     3121 cgtctcgagg cgctgaaaga aaacgggggt gcccgcttgg ctgagtacca cgcgaaagcg
     3181 acagaacacc tgagcacctt gagcgaaaaa gcgaaaccgg cgctggaaga tctacgccag
     3241 ggcttattgc ctgttcttga gagctttaaa gtcagttttc tgtcagctct ggaagaatat
     3301 actaaaaagc tgaataccca gggtaccccc gtgacgcagg aattctggga caacctggaa
     3361 aaagaaaccg agggactgcg tcaggaaatg tccaaagatt tagaagaggt gaaggccaag
     3421 gttcagccat atctcgatga ctttcagaaa aaatggcagg aagagatgga attatatcgt
     3481 caaaaggtgg aaccgctgcg tgcggaactg caagaggggg cacgccaaaa actccatgag
     3541 ctccaagaga agctcagccc attaggcgaa gaaatgcgcg atcgcgcccg tgcacatgtt
     3601 gatgcactcc ggactcattt ggcgccgtat tcggatgaac ttcgccagcg tttggccgca
     3661 cgtctcgagg cgctgaaaga aaacgggggt gcccgcttgg ctgagtacca cgcgaaagcg
     3721 acagaacacc tgagcacctt gagcgaaaaa gcgaaaccgg cgctggaaga tctacgccag
     3781 ggcttattgc ctgttcttga gagctttaaa gtcagttttc tgtcagctct ggaagaatat
     3841 actaaaaagc tgaataccca gtaagcttcc atgggtaccc tcgagcacca ccaccaccac
     3901 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     3961 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     4021 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     4081 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     4141 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4201 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4261 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     4321 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4381 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     4441 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     4501 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4561 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4621 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4681 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4741 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4801 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4861 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4921 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4981 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5041 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5101 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5161 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5221 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5281 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5341 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5401 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5461 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5521 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5581 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5641 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5701 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5761 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     5821 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5881 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5941 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6001 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6061 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6121 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6181 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6241 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6301 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6361 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     6421 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     6481 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6541 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     6601 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     6661 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     6721 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     6781 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     6841 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     6901 ttctg
//
