LOCUS       sequence-495727-        6255 bp    DNA     circular SYN 18-DEC-2025
DEFINITION  Expression of MGYFPDa829 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 6255)
  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 6255)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 18, 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::MGYFPDa829/MGYFPDa829I
FEATURES             Location/Qualifiers
     source          1..6255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(72..1154)
                     /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     1116..1135
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1155..1232)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1438..1457
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1545..1563
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1564..1588
                     /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             1603..1625
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1644..1661
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        2234..2252
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2253..2277
                     /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             2765..2809
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2847..2865)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2861..2908
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2945..3400
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3032..3051)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3242..3263
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3515..4306,4307..4375))
                     /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     4138..4157
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4376..4468)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4549..5137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5038..5057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5291..5308
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5323..5462
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5388..5407)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5548..5570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5564..5755)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
       61 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
      121 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
      181 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
      241 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
      301 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca
      361 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
      421 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
      481 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
      541 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
      601 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
      661 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
      721 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
      781 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
      841 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
      901 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
      961 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     1021 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     1081 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     1141 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     1201 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     1261 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     1321 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     1381 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     1441 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     1501 gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat
     1561 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     1621 ggagatatac catgggcagc agccatcacc atcatcacca cagccaggat ccggacttcc
     1681 acacctatta cgttggcacc gaatccgtac tggttcataa caacggtggt aactgcatga
     1741 aaaccgcgag cgctctggca cagggtggtt ccaaaggcgg ttctacggca atcaacctgc
     1801 cggagtacga tggtaaaacc acgcacggcg ttctggttct ggacaatggt acccaggtgc
     1861 agctggtgtc tggtaatggc gatcctcgct acactaatta tcgcaacaac ggccacgttg
     1921 aacagaaagc tgcgatctac atgcgcgaaa acaacatttc caacgcaact gtttaccaca
     1981 acaataccaa cggcacgtgt ggctactgta acaccatgac tgccaccttt ctgccggaag
     2041 gtgctactct gactgtggtt cctcctaaaa atgccgtcgc aaacaactcc cgcgcgatcg
     2101 catacgttaa aacgtacacc ggcacctcta acgacccgaa aatgtctagc cgttacaaag
     2161 gcaactgagc ggccgcataa tgcttaagtc gaacagaaag taatcgtatt gtacacggcc
     2221 gcataatcga aattaatacg actcactata ggggaattgt gagcggataa caattcccca
     2281 tcttagtata ttagttaagt ataagaagga gatatacata tgctggtcga acactttatg
     2341 ggtcagaaag aatgcgattc cctggaagag ctgcgtgaag tcctgagcga acgtactgaa
     2401 aaaggtgtta acgaatttat catcagcact cacgaacagt tcccttacat gatcatgagc
     2461 gttaaagaaa aatacgcttg tctgtcttac ttccgtgaag aggacgatcc gggttacagc
     2521 tctgtgaacg ctaacccggt tctggacgct gacggtatta gcattttcta taccaacacc
     2581 gattctgaag aaattgaggt ggctaactac agcatcgtca aaatcgaaga cgctgtgagc
     2641 gccgttgagg aattctttga aactctgcag ctgccgaaat gtatcgagtg ggaagaactg
     2701 taaagatctc aattggatat cggccggcca cgcgatcgct gacgtcggta ccctcgagtc
     2761 tggtaaagaa accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc
     2821 agcttaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2881 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     2941 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3001 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3061 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3121 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3181 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3241 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3301 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3361 tttaacgcga attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga
     3421 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3481 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3541 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3601 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3661 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3721 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3781 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3841 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3901 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3961 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4021 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4081 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4141 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4201 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4261 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4321 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4381 tcctttttca atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata
     4441 tttgaatgta tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt
     4501 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4561 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4621 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4681 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4741 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4801 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4861 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4921 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4981 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5041 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5101 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5161 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5221 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     5281 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     5341 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     5401 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     5461 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5521 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5581 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     5641 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     5701 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     5761 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     5821 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     5881 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     5941 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     6001 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     6061 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     6121 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     6181 gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg
     6241 agctgactgg gttga
//
