LOCUS       sequence-410381-        6961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overxpression MBP-TadA8e.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6961)
  AUTHORS   Xiao YL, Wu Y, Tang W
  TITLE     An adenine base editor variant expands context compatibility.
  JOURNAL   Nat Biotechnol. 2024 Jan 2. doi: 10.1038/s41587-023-01994-3.
  PUBMED    38168987
REFERENCE   2  (bases 1 to 6961)
  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 
            Biotechnol. 2024 Jan 2. doi: 10.1038/s41587-023-01994-3."
COMMENT     Sequence Label: pET28a-6xHis-MBP-TEV site-TadA8e
FEATURES             Location/Qualifiers
     source          1..6961
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(884..1966)
                     /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     1928..1947
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1967..2044)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2250..2269
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2353..2372
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2353..2371
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2372..2396
                     /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             2411..2433
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2452..2469
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2479..2496
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2500..3600
                     /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     3574..3597
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3622..3642
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4178..4195
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4248..4266)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4262..4309
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4346..4801
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4433..4452)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4643..4664
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4893..5708)
                     /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(4893..5708)
                     /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     5616..5635
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5700..5719
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5830..6418
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6319..6338
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6572..6589
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6604..6743
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6669..6688)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6829..6851
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(6845..6961,1..75))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
       61 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg tggtaatgat
      121 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
      181 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
      241 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
      301 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
      361 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
      421 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
      481 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
      541 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
      601 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
      661 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
      721 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
      781 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
      841 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
      901 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      961 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     1021 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     1081 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     1141 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     1201 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     1261 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     1321 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     1381 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     1441 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     1501 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     1561 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     1621 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     1681 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     1741 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     1801 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     1861 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     1921 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     1981 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     2041 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     2101 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     2161 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     2221 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     2281 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     2341 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     2401 tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat
     2461 catcatcaca gcagcggcct ggtgccgcgc ggcagccata tgaaaatcga agaaggtaaa
     2521 ctggtaatct ggattaacgg cgataaaggc tataacggac tcgctgaagt cggtaagaaa
     2581 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
     2641 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
     2701 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
     2761 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
     2821 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
     2881 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
     2941 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
     3001 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
     3061 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
     3121 gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
     3181 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
     3241 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
     3301 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
     3361 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
     3421 tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa
     3481 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     3541 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     3601 tctggaggat ctagcggatc cgaaaacctg tacttccagg gaagcggcag cagcggcggc
     3661 agcatgtctg aggtggagtt ttcccacgag tactggatga gacatgccct gaccctggcc
     3721 aagagggcac gcgatgagag ggaggtgcct gtgggagccg tgctggtgct gaacaataga
     3781 gtgatcggcg agggctggaa cagagccatc ggcctgcacg acccaacagc ccatgccgaa
     3841 attatggccc tgagacaggg cggcctggtc atgcagaact acagactgat tgacgccacc
     3901 ctgtacgtga cattcgagcc ttgcgtgatg tgcgccggcg ccatgatcca ctctaggatc
     3961 ggccgcgtgg tgtttggcgt gaggaactca aaacgcggcg ccgcaggctc cctgatgaac
     4021 gtgctgaact accccggcat gaatcaccgc gtcgaaatta ccgagggaat cctggcagat
     4081 gaatgtgccg ccctgctgtg cgatttctat cggatgccta gacaggtgtt caatgctcag
     4141 aagaaggccc agagctccat caactaacta actcgagcat caccaccacc accactgaga
     4201 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     4261 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     4321 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4381 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4441 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4501 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4561 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4621 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4681 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4741 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     4801 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4861 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     4921 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     4981 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     5041 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     5101 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     5161 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     5221 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     5281 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     5341 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     5401 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     5461 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     5521 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     5581 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     5641 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     5701 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     5761 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5821 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5881 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5941 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     6001 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6061 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6121 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6181 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgaaaaa gcgccacgct
     6241 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6301 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6361 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6421 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6481 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6541 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6601 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     6661 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     6721 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     6781 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     6841 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     6901 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     6961 g
//
