LOCUS       sequence-495453-        7383 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Expression of DddA11-MBP and its immunity protein in bacterial cells
            for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7383)
  AUTHORS   Roh H, Shen SP, Hu Y, Kwok HS, Siegenfeld AP, Lee C, Zepeda MU, Guo 
            CJ, Roseman SA, Comenho C, Sankaran VG, Buenrostro JD, Liau BB
  TITLE     Coupling CRISPR scanning with targeted chromatin accessibility 
            profiling using a double-stranded DNA deaminase.
  JOURNAL   Nat Methods. 2025 Oct;22(10):2083-2093. doi: 
            10.1038/s41592-025-02811-2. Epub 2025 Sep 11.
  PUBMED    40935921
REFERENCE   2  (bases 1 to 7383)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-025-02811-2"; journalName: "Nat Methods"; date: 
            "2025-10"; volume: "22"; issue: "10"; pages: "2083-2093"
COMMENT     Sequence Label: pETDuet::dddA11-MBP/dddI
FEATURES             Location/Qualifiers
     source          1..7383
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             154..174
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             196..1293
                     /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="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1150..1159
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1267..1290
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     promoter        1362..1380
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1385..1401
                     /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)."
     CDS             1881..1925
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1963..1981)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1977..2024
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2061..2516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2148..2167)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2358..2379
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2631..3422,3423..3491))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3254..3273
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3492..3584)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3665..4253
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4154..4173
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4407..4424
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4439..4578
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4504..4523)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4664..4686
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4680..4871)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5443..6525)
                     /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     6487..6506
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6526..6603)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6809..6828
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6916..6934
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6935..6959
                     /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             6974..6996
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7015..7032
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gtgaacatga ttgagactct tctgcccgaa aacgccaaga tgaccgttgt tccacctgaa
       61 ggagcgatac cagtcaagcg tggcgcaact ggtgagacaa aagtgtttat tgggaatagc
      121 aattctccga aatcccccac gaaaggaggt tgtgagaacc tgtacttcca gggtggcggt
      181 tccggcggag ggtcgaaaat cgaagaaggt aaactggtaa tctggattaa cggcgataaa
      241 ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc
      301 accgttgagc atccggataa actggaagag aaattcccac aggttgcggc aactggcgat
      361 ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg
      421 ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat
      481 gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg
      541 atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg
      601 gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac
      661 ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag
      721 tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg
      781 gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct
      841 gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc
      901 gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc
      961 aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg
     1021 gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa
     1081 gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggc gaaagatcca
     1141 cgtattgccg ccaccatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag
     1201 atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag
     1261 actgtcgatg aagccctgaa agacgcgcag acttgagcgg ccgcataatg cttaagtcga
     1321 acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg
     1381 gaaattgtga gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga
     1441 tatacatatg tacgcagacg atttcgacgg cgaaattgag atcgatgagg ttgatagctt
     1501 ggtcgagttc ctcagcagaa gacctgcatt tgacgcaaat aattttgttt tgacgtttga
     1561 ggaaagtggt tttcctcaat tgaatatttt tgcaaaaaac gatatcgccg ttgtttatta
     1621 tatggacatc ggcgagaatt tcgtttcgaa aggcaattct gcgtcaggtg gtacggagaa
     1681 gttttacgag aataagcttg gcggggaagt agacctgtcg aaagattgcg ttgtgagtaa
     1741 agagcaaatg attgaagcgg caaagcagtt ttttgctacg aagcaacggc ccgagcaact
     1801 gacatggagc gagttgtaaa gatctcaatt ggatatcggc cggccacgcg atcgctgacg
     1861 tcggtaccct cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg
     1921 actcgtctac tagcgcagct taattaacct aggctgctgc caccgctgag caataactag
     1981 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     2041 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2101 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2161 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2221 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2281 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2341 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2401 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2461 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttctgg
     2521 cggcacgatg gcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2581 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2641 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2701 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2761 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2821 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2881 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2941 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3001 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3061 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3121 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3181 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3241 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3301 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3361 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3421 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3481 tgaatactca tactcttcct ttttcaatca tgattgaagc atttatcagg gttattgtct
     3541 catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggtc atgaccaaaa
     3601 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3661 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3721 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3781 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3841 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3901 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3961 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4021 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4081 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4141 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4201 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4261 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4321 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4381 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4441 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     4501 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     4561 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4621 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4681 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4741 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4801 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4861 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4921 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4981 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     5041 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     5101 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     5161 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     5221 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     5281 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgct agtcatgccc
     5341 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     5401 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     5461 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5521 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     5581 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     5641 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     5701 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
     5761 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     5821 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     5881 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5941 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     6001 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     6061 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     6121 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     6181 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     6241 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     6301 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     6361 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     6421 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     6481 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     6541 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     6601 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     6661 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     6721 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     6781 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     6841 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatcgatc
     6901 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     6961 ctctagaaat aattttgttt aactttaaga aggagatata ccatgggcag cagccatcac
     7021 catcatcacc acagccagga tccgatagga ctcaacggtg gggcgaacgt ctatcattac
     7081 gcacctaacc cggttggatg ggtcgatccg tggggtttgg ccggcagtta cgcgcttggc
     7141 ccctatcaga tttccgctcc gcagttgccg gcatataatg gacagacggt cggaacgttc
     7201 tactacgtga atgacgcagg tggccttgaa tccaaggtgt tcatttctgg cggacctact
     7261 ccatacccca actacgtctc tgccgggcac gttgaaggtc agtccgcctt atttatgcgc
     7321 gataacggaa tttctgaagg tttggtattc cacaataacc ctaaaggaac gtgcgggttc
     7381 tgc
//
