LOCUS       sequence-273408-        6833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of A3A with N-terminal MBP an C-terminal His 
            tag, both cleavable with TEV protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6833)
  AUTHORS   Schutsky EK, DeNizio JE, Hu P, Liu MY, Nabel CS, Fabyanic EB, Hwang 
            Y, Bushman FD, Wu H, Kohli RM
  TITLE     Nondestructive, base-resolution sequencing of 
            5-hydroxymethylcytosine using a DNA deaminase.
  JOURNAL   Nat Biotechnol. 2018 Oct 8. pii: nbt.4204. doi: 10.1038/nbt.4204.
  PUBMED    30295673
REFERENCE   2  (bases 1 to 6833)
  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. 2018 Oct 8. pii: nbt.4204. doi: 10.1038/nbt.4204."
COMMENT     Sequence Label: pET41-MBP-t-A3A-t-His
FEATURES             Location/Qualifiers
     source          1..6833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             89..1189
                     /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"
     regulatory      1046..1055
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1163..1186
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1238..1249
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1262..1282
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1289..1885
                     /codon_start=1
                     /gene="human APOBEC3A"
                     /gene_synonym="A3A"
                     /product="human cytidine deaminase (C to U editing enzyme) 
                     that efficiently edits methylated cytosines (Wang et al., 
                     2018)"
                     /label=APOBEC3A
                     /note="also known as hA3A"
                     /translation="MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTS
                     VKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSW
                     GCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWD
                     TFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN"
     CDS             1892..1912
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1925..1948
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2001..2019)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2015..2062
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2099..2554
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2186..2205)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2396..2417
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2646..3461)
                     /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(2646..3461)
                     /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     3369..3388
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3453..3472
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3583..4171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4072..4091
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4325..4342
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4357..4496
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4422..4441)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4582..4604
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4598..4789)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5361..6443)
                     /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     6405..6424
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6444..6521)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6727..6746
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gaaaatcgaa gaaggtaaac tggtaatctg
      121 gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga
      181 taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt
      241 tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta
      301 cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta
      361 tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt
      421 tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga
      481 agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa
      541 cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa
      601 gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc
      661 gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta
      721 ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg
      781 ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt
      841 caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag
      901 tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct
      961 ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga
     1021 gttggcgaaa gatccacgta ttgccgccac catggaaaac gcccagaaag gtgaaatcat
     1081 gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc
     1141 cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta attcgagctc
     1201 gaacaacaac aacaataaca ataacaacaa cctcgggatc gagggaagga tttcagaatt
     1261 cgaaaacctg tattttcagg ggtcgacaat ggaagccagc ccagcatccg ggcccagaca
     1321 cttgatggat ccacacatat tcacttccaa ctttaacaat ggcattggaa ggcataagac
     1381 ctacctgtgc tacgaagtgg agcgcctgga caatggcacc tcggtcaaga tggaccagca
     1441 caggggcttt ctacacaacc aggctaagaa tcttctctgt ggcttttacg gccgccatgc
     1501 ggagctgcgc ttcttggacc tggttccttc tttgcagttg gacccggccc agatctacag
     1561 ggtcacttgg ttcatctcct ggagcccctg cttctcctgg ggctgtgccg gggaagtgcg
     1621 tgcgttcctt caggagaaca cacacgtgag actgcgtatc ttcgctgccc gcatctatga
     1681 ttacgacccc ctatataagg aggcactgca aatgctgcgg gatgctgggg cccaagtctc
     1741 catcatgacc tacgatgaat ttaagcactg ctgggacacc tttgtggacc accagggatg
     1801 tcccttccag ccctgggatg gactagatga gcacagccaa gccctgagtg ggaggctgcg
     1861 ggccattctc cagaatcagg gaaacggtac cgaaaacctg tattttcagg gctcgacaat
     1921 ggatcaccac caccaccacc accaccacta acctaggctg ctaaacaaag cccgaaagga
     1981 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     2041 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     2101 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2161 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2221 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2281 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2341 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2401 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2461 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2521 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     2581 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     2641 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2701 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2761 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2821 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2881 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2941 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     3001 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     3061 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     3121 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     3181 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     3241 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     3301 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     3361 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3421 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     3481 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     3541 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3601 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3661 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3721 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3781 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3841 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3901 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3961 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4021 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4081 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4141 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4201 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4261 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4321 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     4381 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     4441 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     4501 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4561 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4621 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     4681 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4741 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4801 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4861 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4921 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4981 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     5041 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     5101 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     5161 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     5221 tcctcaacga caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga
     5281 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5341 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5401 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5461 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5521 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5581 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5641 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5701 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5761 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5821 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5881 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5941 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     6001 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     6061 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     6121 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     6181 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     6241 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     6301 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     6361 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     6421 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6481 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6541 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6601 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6661 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6721 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6781 ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga aat
//
