LOCUS       sequence-368328-        7105 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression human APOBEC3A in E.coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7105)
  AUTHORS   Fabyanic EB, Hu P, Qiu Q, Berrios KN, Connolly DR, Wang T, Flournoy 
            J, Zhou Z, Kohli RM, Wu H
  TITLE     Joint single-cell profiling resolves 5mC and 5hmC and reveals their 
            distinct gene regulatory effects.
  JOURNAL   Nat Biotechnol. 2023 Aug 28. doi: 10.1038/s41587-023-01909-2.
  PUBMED    37640946
REFERENCE   2  (bases 1 to 7105)
  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. 2023 Aug 28. doi: 10.1038/s41587-023-01909-2."
COMMENT     Sequence Label: pET-His6MBP-A3A-Gly6His6
FEATURES             Location/Qualifiers
     source          1..7105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     658..677
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(697..774)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     980..999
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1083..1102
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1083..1101
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1102..1126
                     /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             1141..1163
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1182..1199
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1209..2309
                     /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     2283..2306
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2364..2387
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2994..3017
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3051..3068
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3121..3139)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3135..3182
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3219..3674
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3306..3325)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3516..3537
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3767..4582)
                     /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(3767..4582)
                     /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     4490..4509
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4574..4593
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4704..5292
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5193..5212
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5446..5463
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5478..5617
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5543..5562)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5703..5725
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5719..5910)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6719..7105,1..696))
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPXTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
       61 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
      121 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
      181 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
      241 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
      301 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
      361 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
      421 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
      481 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
      541 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
      601 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
      661 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
      721 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
      781 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
      841 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
      901 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
      961 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     1021 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     1081 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     1141 tttgtttaac tttaagaagg agatatacca tgggttcttc tcaccatcac catcaccatg
     1201 gttcttctat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
     1261 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
     1321 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
     1381 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
     1441 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
     1501 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
     1561 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
     1621 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
     1681 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
     1741 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
     1801 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
     1861 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
     1921 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
     1981 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     2041 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     2101 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     2161 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     2221 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     2281 tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac aacaataaca
     2341 ataacaacaa cctcgggatc gagctggaag tgctgttcca ggggccgaat atggaggcgt
     2401 cgcctgcatc ggggcctcgt catcttatgg accctcatat ctttacaagc aattttaaca
     2461 atggaatcgg ccgtcacaaa acgtaccttt gctatgaagt tgaacgtctg gataacggga
     2521 catccgtaaa aatggatcag catcgcggct tcctgcataa tcaggccaaa aacttgttat
     2581 gcggttttta tgggcgccat gcagagttac gtttcttaga tctggtaccc tctcttcagt
     2641 tagatccggc acagatttac cgcgttactt ggttcattag ctggagtcct tgtttttcct
     2701 gggggtgcgc tggcgaggtc cgtgcatttt tgcaagaaaa cacccatgtg cgtcttcgta
     2761 tttttgccgc ccgtatttat gattacgacc ctctgtataa ggaagccctg cagatgttac
     2821 gcgacgcagg cgctcaagtg tcgattatga cgtacgacga gttcaagcat tgttgggata
     2881 cgtttgttga ccaccagggt tgtcccttcc agccgtggga cggtttagac gaacattcgc
     2941 aggccctgag cggtcgttta cgtgccattc tgcagaacca ggggaatgga tccctggaag
     3001 ttctgttcca ggggcccgga actcgaggag gaggaggagg agggctcgag caccaccacc
     3061 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     3121 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     3181 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     3241 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3301 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3361 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3421 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3481 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3541 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3601 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaactagt
     3661 aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3721 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     3781 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     3841 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     3901 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     3961 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     4021 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     4081 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     4141 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     4201 ggaacactgc cagcgcatca acaatgtttt cacctgaatc aggatattct tctaatacct
     4261 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     4321 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     4381 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     4441 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     4501 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     4561 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     4621 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4681 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4741 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4801 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     4861 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4921 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4981 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5041 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5101 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5161 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5221 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5281 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5341 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5401 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     5461 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5521 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     5581 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     5641 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5701 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     5761 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     5821 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5881 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5941 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     6001 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     6061 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     6121 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     6181 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     6241 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     6301 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     6361 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     6421 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     6481 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     6541 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     6601 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     6661 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     6721 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6781 cgcggggaga ggcggtttgc gtawkgggcg ccagggtggt ttttcttttc accagtgaga
     6841 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     6901 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     6961 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     7021 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     7081 cagtgggaac gataccctca ttcag
//
