LOCUS       sequence-426479-        7491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express fusion proteins of Protein A/G and MMLV reverse 
            transcriptase (25-497) in bacterial systems.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7491)
  AUTHORS   Xiao Y, Chen YM, Zou Z, Ye C, Dou X, Wu J, Liu C, Liu S, Yan H, Wang
            P, Zeng TB, Liu Q, Fei J, Tang W, He C
  TITLE     Profiling of RNA-binding protein binding sites by in situ reverse 
            transcription-based sequencing.
  JOURNAL   Nat Methods. 2024 Jan 10. doi: 10.1038/s41592-023-02146-w.
  PUBMED    38200227
REFERENCE   2  (bases 1 to 7491)
  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 
            Methods. 2024 Jan 10. doi: 10.1038/s41592-023-02146-w."
COMMENT     Sequence Label: His6-pAG-RTase
FEATURES             Location/Qualifiers
     source          1..7491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             316..525
                     /codon_start=1
                     /product="immunoglobulin binding domain of Streptoccocus 
                     protein G"
                     /label=protein G
                     /translation="TTYKLVINGKTLKGETTTEAVDAETAERHFKQYANDNGVDGEWTY
                     DDATKTFTVTEKPEVIDASELTPAV"
     CDS             2044..2061
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2114..2132)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2128..2175
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2212..2667
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2299..2318)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2509..2530
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2759..3574)
                     /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(2759..3574)
                     /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     3482..3501
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3566..3585
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3696..4284
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4185..4204
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4438..4455
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4470..4609
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4535..4554)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4695..4717
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4711..4902)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5711..6793)
                     /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     6755..6774
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6794..6871)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7077..7096
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7180..7199
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7180..7198
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7199..7223
                     /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             7238..7260
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7279..7296
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7306..7323
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             7327..7359
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(7381..7491,1..315)
                     /codon_start=1
                     /product="immunoglobulin binding domains of Staphylocuccus 
                     aureus protein A"
                     /label=protein A
                     /translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL
                     PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP
                     NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK"
ORIGIN
        1 gctttctatg aaatcttaca tttacctaac ttaaacgaag aacaacgcaa tggtttcatc
       61 caaagcctaa aagatgaccc aagccaaagc gctaaccttt tagcagaagc taaaaagcta
      121 aatgatgctc aagcaccaaa agctgacaac aaattcaaca aagaacaaca aaatgctttc
      181 tatgaaattt tacatttacc taacttaact gaagaacaac gtaacggctt catccaaagc
      241 cttaaagacg atccttcagt gagcaaagaa attttagcag aagctaaaaa gctaaacgat
      301 gctcaagcac caaaaacaac ttataaatta gtcatcaacg ggaaaacgct gaagggtgaa
      361 accacgacag aggccgtaga tgcggagaca gcggagcgcc actttaagca atacgcgaat
      421 gataacggtg tagacggcga gtggacctac gacgacgcga caaagacctt taccgtcacg
      481 gagaaacctg aggttatcga cgcgtctgag ttgacgccag ccgtatctag cggaggatcc
      541 tctggcagcg agacaccagg aacaagcgag tcagcaacac cagagagcag tggcggcagc
      601 agcggcggca gcagctggct gtctgatttt cctcaggcct gggcggaaac cgggggcatg
      661 ggactggcag ttcgccaagc tcctctgatc atacctctga aagcaacctc tacccccgtg
      721 tccataaaac aataccccat gtcacaagaa gccagactgg ggatcaagcc ccacatacag
      781 agactgttgg accagggaat actggtaccc tgccagtccc cctggaacac gcccctgcta
      841 cccgttaaga aaccagggac taatgattat aggcctgtcc aggatctgag agaagtcaac
      901 aagcgggtgg aagacatcca ccccaccgtg cccaaccctt acaacctctt gagcgggctc
      961 ccaccgtccc accagtggta cactgtgctt gatttaaagg atgccttttt ctgcctgaga
     1021 ctccacccca ccagtcagcc tctcttcgcc tttgagtgga gagatccaga gatgggaatc
     1081 tcaggacaat tgacctggac cagactccca cagggtttca aaaacagtcc caccctgttt
     1141 aatgaggcac tgcacagaga cctagcagac ttccggatcc agcacccaga cttgatcctg
     1201 ctacagtacg tggatgactt actgctggcc gccacttctg agctagactg ccaacaaggt
     1261 actcgggccc tgttacaaac cctagggaac ctcgggtatc gggcctcggc caagaaagcc
     1321 caaatttgcc agaaacaggt caagtatctg gggtatcttc taaaagaggg tcagagatgg
     1381 ctgactgagg ccagaaaaga gactgtgatg gggcagccta ctccgaagac ccctcgacaa
     1441 ctaagggagt tcctagggaa ggcaggcttc tgtcgcctct tcatccctgg gtttgcagaa
     1501 atggcagccc ccctgtaccc tctcaccaaa ccggggactc tgtttaattg gggcccagac
     1561 caacaaaagg cctatcaaga aatcaagcaa gctcttctaa ctgccccagc cctggggttg
     1621 ccagatttga ctaagccctt tgaactcttt gtcgacgaga agcagggcta cgccaaaggt
     1681 gtcctaacgc aaaaactggg accttggcgt cggccggtgg cctacctgtc caaaaagcta
     1741 gacccagtag cagctgggtg gcccccttgc ctacggatgg tagcagccat tgccgtactg
     1801 acaaaggatg caggcaagct aaccatggga cagccactag tcattctggc cccccatgca
     1861 gtagaggcac tagtcaaaca accccccgac cgctggcttt ccaacgcccg gatgactcac
     1921 tatcaggcct tgcttttgga cacggaccgg gtccagttcg gaccggtggt agccctgaac
     1981 ccggctacgc tgctcccact gcctgaggaa gggctgcaac acaactgcct tgattaactc
     2041 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     2101 ttggctgctg ccgccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     2161 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     2221 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2281 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2341 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2401 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2461 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2521 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2581 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2641 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     2701 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     2761 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     2821 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     2881 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     2941 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     3001 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     3061 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     3121 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     3181 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     3241 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     3301 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     3361 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     3421 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     3481 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     3541 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     3601 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     3661 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3721 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3781 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3841 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3901 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3961 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4021 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4081 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4141 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4201 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4261 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4321 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4381 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4441 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     4501 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4561 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     4621 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4681 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4741 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     4801 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4861 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4921 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4981 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     5041 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     5101 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     5161 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     5221 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     5281 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     5341 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     5401 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     5461 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     5521 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     5581 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     5641 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     5701 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5761 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     5821 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     5881 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     5941 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     6001 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     6061 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     6121 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     6181 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     6241 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     6301 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     6361 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     6421 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     6481 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     6541 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     6601 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     6661 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     6721 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     6781 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     6841 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     6901 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     6961 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     7021 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     7081 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     7141 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     7201 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
     7261 tataccatgg gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc
     7321 agccatatgg ctagcatgac tggtggacag caaatgggtc gcggatccat gattacgcca
     7381 agcttaaaag atgacccaag ccaaagtgct aacctattgt cagaagctaa aaagttaaat
     7441 gaatctcaag caccgaaagc ggataacaaa ttcaacaaag aacaacaaaa t
//
