LOCUS       sequence-436661-        6780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector to purify BS2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6780)
  AUTHORS   Pani S, Qiu T, Kentala K, Azizi SA, Dickinson BC
  TITLE     Bioorthogonal masked acylating agents for proximity-dependent RNA 
            labelling.
  JOURNAL   Nat Chem. 2024 Apr 9. doi: 10.1038/s41557-024-01493-1.
  PUBMED    38594368
REFERENCE   2  (bases 1 to 6780)
  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: "Nat Chem. 
            2024 Apr 9. doi: 10.1038/s41557-024-01493-1."
COMMENT     Sequence Label: Pet28a-His-tag-BS2
FEATURES             Location/Qualifiers
     source          1..6780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             230..247
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(300..318)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      314..361
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      398..853
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(485..504)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     695..716
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(945..1760)
                     /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(945..1760)
                     /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     1668..1687
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1752..1771
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1882..2470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2371..2390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2624..2641
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2656..2795
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2721..2740)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2881..2903
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2897..3088)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3897..4979)
                     /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     4941..4960
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4980..5057)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5263..5282
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5366..5385
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5366..5384
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5385..5409
                     /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             5424..5446
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5465..5482
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5492..5509
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 tgaggtgaaa cagctttctc acacgataca atcagcgtgg atcacgttcg ccaaaacagg
       61 aaacccaagc accgaagctg tgaattggcc tgcgtatcat gaagaaacga gagagacgct
      121 gattttagac tcagagatta cgatcgaaaa cgatcccgaa tctgaaaaaa ggcagaagct
      181 attcccttca aaaggagaag gatcctgaaa gcttgcggcc gcactcgagc accaccacca
      241 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
      301 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
      361 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
      421 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
      481 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
      541 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
      601 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
      661 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
      721 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
      781 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
      841 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
      901 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
      961 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     1021 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     1081 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     1141 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     1201 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     1261 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     1321 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     1381 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     1441 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     1501 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     1561 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     1621 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     1681 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     1741 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     1801 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1861 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1921 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1981 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2041 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2101 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2161 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2221 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2281 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2341 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2401 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2461 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     2521 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     2581 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     2641 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2701 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     2761 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     2821 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     2881 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     2941 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3001 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3061 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3121 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     3181 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     3241 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     3301 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     3361 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     3421 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     3481 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     3541 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     3601 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     3661 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     3721 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     3781 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     3841 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     3901 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3961 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4021 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4081 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4141 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4201 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     4261 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     4321 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     4381 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     4441 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     4501 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     4561 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     4621 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     4681 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     4741 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     4801 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     4861 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     4921 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     4981 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5041 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5101 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5161 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     5221 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     5281 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     5341 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     5401 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata ccatgggcag
     5461 cagccatcat catcatcatc acagcagcgg cctggtgccg cgcggcagcc atatgactca
     5521 tcaaatagta acgactcaat acggcaaagt aaaaggcaca acggaaaacg gcgtacataa
     5581 gtggaaaggc atcccctatg ccaagccgcc tgtcggacaa tggcgtttta aagcacctga
     5641 gccgcctgaa gtgtgggaag atgtgcttga tgccacagcg tacggctcta tttgcccgca
     5701 gccgtctgat ttgctgtcac tttcgtatac tgagctgccc cgccagtccg aggattgctt
     5761 gtatgtcaat gtatttgcgc ctgacacccc aagtaaaaat cttcctgtca tggtgtggat
     5821 tcacggaggc gctttttatc taggagcggg cagtgagcca ttgtatgacg gatcaaaact
     5881 tgcggcacag ggagaagtca ttgtcgttac attgaactat cggctggggc cgtttggctt
     5941 tttgcacttg tcttcattta atgaggcgta ttctgataac cttgggcttt tagaccaagc
     6001 cgccgcgctg aaatgggtgc gagagaatat ttcagcgttt ggcggtgatc ccgataacgt
     6061 aacagtattt ggagaatccg ccggcgggat gagcattgcc gcgctgcttg ctatgcctgc
     6121 ggcaaaaggc ctgttccaga aagcaatcat ggaaagcggc gcttctcgaa cgatgacgaa
     6181 agaacaagcg gcgagcacct cggcagcctt tttacaggtc cttgggatta acgagggcca
     6241 actggataaa ttgcatacgg tttctgcgga agatttgcta aaagcggctg atcagcttcg
     6301 gattgcagaa aaagaaaata tctttcagct gttcttccag cccgcccttg atccgaaaac
     6361 gctgcctgaa gaaccagaaa aagcgatcgc agaaggggct gcttccggta ttccgctatt
     6421 aattggaaca acccgtgatg aaggatattt atttttcacc ccggattcag acgttcattc
     6481 tcaggaaacg cttgatgcag cgctcgagta tttactaggg aagccgctgg cagagaaagt
     6541 tgccgatttg tatccgcgtt ctctggaaag ccaaattcac atgatgactg atttattatt
     6601 ttggcgccct gccgtcgcct atgcatccgc acagtctcat tacgcccctg tctggatgta
     6661 caggttcgat tggcacccga agaagccgcc gtacaataaa gcgtttcacg cattagagct
     6721 tccttttgtc tttggaaatc tggacggatt ggaacgaatg gcaaaagcgg agattacgga
//
