LOCUS       sequence-444232-        7650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for bacterial purification of codon-optimized TadCBE-Cas9NG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7650)
  AUTHORS   Martin-Rufino JD, Caulier A, Lee S, Castano N, King E, Joubran S, 
            Jones M, Goldman SR, Arora UP, Wahlster L, Lander ES, Sankaran VG
  TITLE     Transcription factor networks disproportionately enrich for 
            heritability of blood cell phenotypes.
  JOURNAL   Science. 2025 Apr 4;388(6742):52-59. doi: 10.1126/science.ads7951. 
            Epub 2025 Apr 3.
  PUBMED    40179192
REFERENCE   2  (bases 1 to 7650)
  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"; doi: 
            "10.1126/science.ads7951"; journalName: "Science"; date: "2025-04-4-
            4"; volume: "388"; issue: "6742"; pages: "52-59"
COMMENT     Sequence Label: TadCBE-Cas9NG
FEATURES             Location/Qualifiers
     source          1..7650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1336..1584
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             1615..1863
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             1909..1929
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1944..1962)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1958..2005
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2103..2132
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      2162..2707
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     2608..2627
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2824..2841
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2972..2999)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3023..3832)
                     /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="MSNIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3746..3765
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3833..3923)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(3932..3962)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        4027..4145
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             4180..4203
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             4237..4257
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 attctggaca gccgcatgaa tactaagtac gatgaaaatg ataaactcat tcgtgaggtc
       61 aaggtcatta ccctgaagtc taagctcgtc agcgattttc gtaaagactt ccagttctat
      121 aaggtgcgcg aaatcaacaa ttaccaccac gcccatgatg catatctgaa cgccgtggtg
      181 ggtacggctc tgatcaagaa atatccgaaa ctggagtccg agtttgtcta tggtgactac
      241 aaagtgtatg acgtccgcaa aatgatcgcc aaatcggaac aagagattgg caaggcaacc
      301 gcgaagtact ttttctattc caacattatg aactttttca agaccgagat caccctggca
      361 aacggtgaga tccgtaaacg tccgctgatc gaaactaacg gtgaaaccgg tgaaatcgtg
      421 tgggacaaag gtcgtgactt tgcaaccgtg cgtaaagtgc tgtcgatgcc acaggtcaac
      481 atcgttaaga aaaccgaagt tcaaactggt ggctttagca aggagagcat tcgtcctaag
      541 cgcaacagcg acaagctgat tgcacgtaag aaggactggg acccgaagaa gtacggcggc
      601 tttgtgtccc cgacggttgc gtatagcgtt ctcgttgtgg cgaaagtcga gaaaggcaag
      661 agcaaaaagc tgaaaagcgt taaagaattg ctgggtatca cgattatgga gcgtagcagc
      721 ttcgaaaaga atcctattga tttcttggaa gcaaaaggtt acaaggaggt gaagaaagac
      781 ttgatcatca aactgccgaa gtattccctg ttcgagctgg aaaacggccg caaacgtatg
      841 ctggccagcg ctcgctttct gcaaaagggc aacgaactgg ctctgccgag caaatatgtg
      901 aatttcctgt acctggccag ccactacgag aaactgaagg gctccccgga agataacgag
      961 cagaagcagc tgttcgttga gcagcacaag cattacctgg acgagattat cgagcagatc
     1021 agcgagttta gcaaacgcgt gattctggcg gatgcgaatt tggataaagt tctgtcggcc
     1081 tataataaac accgcgacaa accgattcgc gagcaggcgg agaacattat ccacttgttc
     1141 accctgacca atctgggtgc tccgcgtgct ttcaaatact ttgataccac gattgatcgc
     1201 aaagtgtatc gttccaccaa agaggtactg gacgcgaccc tgatccacca gagcatcacc
     1261 ggcttgtacg aaacgcgcat cgatctgtct caactgggcg gtgacagcgg tggttcgggt
     1321 ggtagcggtg gcagcaccaa cctgagcgac attattgaaa aagaaaccgg caaacaactg
     1381 gtcatccaag aaagcattct gatgttgccg gaggaagttg aagaggtgat tggcaataaa
     1441 ccggagagcg atatcctggt gcataccgcg tacgacgaaa gcactgatga gaatgtgatg
     1501 ttgctgacct ccgatgcacc ggagtacaaa ccgtgggcgt tggtgatcca agatagcaat
     1561 ggtgaaaaca agattaagat gctgtccggt ggttccggtg gctctggtgg ttccaccaac
     1621 ttgtccgaca tcattgagaa agaaaccggt aagcagctgg ttattcaaga atctattctg
     1681 atgctgccgg aagaggttga agaagtcatc ggcaataagc ctgagagcga cattttggtt
     1741 cataccgctt acgatgagag cacggatgaa aatgtaatgc tgctgacgag cgacgctccg
     1801 gagtacaagc cgtgggcact ggtgattcaa gatagcaacg gcgagaataa gatcaaaatg
     1861 ctgagcggtg gtagcaaacg cacagccgat ggatcagaat ttgaatctcc gaaaaagaaa
     1921 cgcaaggtgt aaagcggccg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1981 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccgggt aacgaattca
     2041 agcttgatat cattcaggac gagcctcaga ctccagcgta actggactgc aatcaactca
     2101 ctggctcacc ttcacgggtg ggcctttctt cggtagaagt cttcttaata agatgatctt
     2161 cttgagatcg ttttggtctg cgcgtaatct cttgctctga aaacgaaaaa accgccttgc
     2221 agggcggttt ttcgaaggtt ctctgagcta ccaactcttt gaaccgaggt aactggcttg
     2281 gaggagcgca gtcaccaaaa cttgtccttt cagtttagcc ttaaccggcg catgacttca
     2341 agactaactc ctctaaatca attaccagtg gctgctgcca gtggtgcttt tgcatgtctt
     2401 tccgggttgg actcaagacg atagttaccg gataaggcgc agcggtcgga ctgaacgggg
     2461 ggttcgtgca tacagtccag cttggagcga actgcctacc cggaactgag tgtcaggcgt
     2521 ggaatgagac aaacgcggcc ataacagcgg aatgacaccg gtaaaccgaa aggcaggaac
     2581 aggagagcgc acgagggagc cgccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2641 gtttcgccac cactgatttg agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct
     2701 atggaaaaac ggctttgccg cggccctctc acttccctgt taagtatctt cctggcatct
     2761 tccaggaaat ctccgccccg ttcgtaagcc atttccgctc gccgcagtcg aacgaccgag
     2821 cgtagcgagt cagtgagcga ggaagcggaa tatatcctgt atcacatatt ctgctgacgc
     2881 accggtgcag ccttttttct cctgccacat gaagcacttc actgacaccc tcatcagtgc
     2941 caacatagta agccagtata cactccgcta gcgcagaaag gcccacccga aggtgagcca
     3001 ggtgattaca tttgggccct cattagaaaa actcatcgag catcaaatga aattgcaatt
     3061 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3121 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3181 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3241 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3301 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3361 aaccgttatt cattcgtgat tgcgcctgag cgaggcgaaa tacgcgatcg ctgttaaaag
     3421 gacaattaca aacaggaatc gagtgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3481 tattttcacc tgaatcagga tattcttcta atacctggaa cgctgttttt ccggggatcg
     3541 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagtg
     3601 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3661 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aagcgataga
     3721 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3781 ccatgttgga atttaatcgc ggcctcgacg tttcccgttg aatattgctc atagctcctg
     3841 aaaatctcga taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt
     3901 tggaacctct tacgtgccga tcaagaagac ggtcaaaagc ctccggtcgg aggcttttga
     3961 ctttctgcta tggaggtcag gtatgattta aatggtcagt attgagcgat atctagagaa
     4021 ttcgtccacc acaattcagc aaattgtgaa catcatcacg ttcatctttc cctggttgcc
     4081 aatggcccat tttcctgtca gtaacgagaa ggtcgcgaat tcaggcgctt tttagactgg
     4141 tcgtaatgaa attcttttta agaaggagat atacatatgc accatcacca tcatcatcat
     4201 cacaaacgta ccgcagatgg tagcgaattt gaaagcccga aaaaaaagcg taaagttagt
     4261 agcgaagttg aatttagcca cgaatactgg atgcgtcatg cgttgacgct ggcgaagcgt
     4321 gcgcgtgacg aacgcaaggc gccggtgggt gctgttctgg tgctgaacaa tcgtgtaatc
     4381 ggtgaaggtt ggaatcgcgc gatcggtctg catgatccaa ccgcgcacgc agaaatcata
     4441 gcgctgcgtc agggtggcct ggtgatgcaa aattaccgtc tgatcgatgc gacgctgtat
     4501 gtgaccttcg aaccgtgtgt catgtgtgcc ggtgccatga tcaacagccg cattggccgt
     4561 gtggtctttg gcgttcgtaa cagcaaacgt ggcgcagcag gctctctgat gaacgtcctg
     4621 aattacccgg gtatgaacca tcgtgttgag attacggagg gcatcttagc ggatgaatgt
     4681 gctgcgctgc tgtgcgattt ctatcgcatg ccgcgtcaag tctttaacgc acagaagaag
     4741 gcacaatcta gcattaactc tggcggttcg tctggtggta gcagcggcag cgaaacgccg
     4801 ggtaccagcg agagcgcaac cccggaaagc tccggtggca gctctggcgg ctctgacaaa
     4861 aagtattcga ttggcctggc gatcggcacg aacagcgtcg gttgggctgt tattaccgac
     4921 gagtataaag tgccgagcaa gaagtttaaa gtcttgggta acaccgatcg ccatagcatc
     4981 aaaaagaatc tgattggtgc cctgctgttt gatagcggcg aaacggcaga agcgacccgt
     5041 ctgaaacgta ccgcacgtcg tcgctacact cgtcgtaaaa accgcatttg ctatttgcaa
     5101 gagatcttca gcaatgaaat ggccaaagtt gacgacagct tctttcaccg tctggaggag
     5161 agctttctgg ttgaagagga taagaaacac gagcgtcacc cgatctttgg taatatcgtt
     5221 gacgaggtcg cgtatcacga gaagtacccg acgatctacc acttgcgtaa gaaactggtg
     5281 gacagcaccg ataaagccga tctgcgtctg atttacttgg cgttggcgca catgatcaag
     5341 ttccgcggtc atttcctgat cgagggtgac ctgaacccag ataattccga cgtcgacaag
     5401 ctgttcatcc agctggttca gacctacaac caattgtttg aagaaaaccc gattaatgca
     5461 tctggtgtcg acgcgaaggc gattctgagc gcgcgtctga gcaagtctcg tcgtctggag
     5521 aatctgatcg cgcaattgcc gggtgagaag aaaaatggtt tgtttggcaa cttgatcgcg
     5581 ctgagcctgg gtctgacccc gaactttaag agcaatttcg acctggcaga agatgcgaaa
     5641 ctgcagctga gcaaagacac ctatgatgac gacctggata atctcctggc acaaattggt
     5701 gaccagtatg cggacctgtt cctggctgcg aaaaatctgt cggacgccat tctgctgtcg
     5761 gacattctgc gcgttaacac tgagatcacg aaagcaccgc tgtctgcgag catgattaaa
     5821 cgttatgacg agcaccatca agacctgacc ctgctgaagg ccctggtccg tcagcaactg
     5881 ccggagaagt ataaagaaat ttttttcgat cagagcaaga acggttacgc aggctatatc
     5941 gatggcggtg cgagccaaga agagttttac aaattcatta aaccgatttt ggagaagatg
     6001 gacggtaccg aggagttgct ggtgaaactg aatcgcgaag atctgctgcg caaacaacgt
     6061 acgttcgata acggtagcat cccacaccag attcacctgg gtgagctgca cgcgattctg
     6121 cgtcgtcagg aagatttcta cccattcctg aaagataatc gtgaaaagat tgagaaaatc
     6181 ttgacgttcc gcattccgta ctacgtcggt ccgctggcac gtggcaacag ccgtttcgct
     6241 tggatgacgc gcaagagcga ggaaactatc acgccgtgga actttgagga agtcgtcgac
     6301 aaaggtgcga gcgcgcaaag ctttattgag cgcatgacca actttgacaa gaatctgccg
     6361 aacgagaaag tcttgccaaa gcacagcctg ctgtacgagt atttcacggt gtacaatgaa
     6421 ctgaccaaag ttaagtatgt caccgagggt atgcgtaaac cagcgttcct gtccggtgaa
     6481 cagaagaaag caattgtgga tctgttgttt aagacgaacc gtaaagtcac ggtgaagcag
     6541 ttgaaagagg attacttcaa aaagatcgaa tgctttgaca gcgtggaaat tagcggtgtt
     6601 gaggaccgtt tcaacgccag cttgggcacc taccatgatc tgctgaaaat cattaaggac
     6661 aaggacttcc tggataacga agaaaacgag gatattctgg aggatattgt cctgacgctg
     6721 accctcttcg aagatcgcga aatgattgaa gagagactga aaacttacgc ccacttgttt
     6781 gacgataaag tcatgaaaca actgaagcgc cgtcgttaca ccggttgggg tcgtctgagc
     6841 cgtaagctga tcaacggtat ccgtgataag caatctggca aaacgatcct ggactttctg
     6901 aaaagcgatg gtttcgcgaa ccgcaacttc atgcagctta ttcacgacga ttctctgacc
     6961 tttaaggagg acatccaaaa agcccaggtt agcggtcagg gcgattccct gcatgagcat
     7021 attgctaact tagcgggtag cccggcaatc aaaaagggca ttctgcaaac cgttaaggtt
     7081 gtggacgagt tggttaaggt catgggccgt cataagccgg agaatatcgt tattgagatg
     7141 gcgcgcgaga atcagaccac gcagaaaggc caaaagaata gccgtgaacg tatgaaacgt
     7201 attgaagagg gtatcaaaga gctgggcagc caaatcttga aggaacatcc ggtggagaac
     7261 acgcagctgc agaatgaaaa gctgtacctg tactatctgc aaaacggtcg tgatatgtac
     7321 gttgaccaag agctggacat caaccgcctg agcgattatg atgttgatca cattgtgccg
     7381 caatcttttc tgaaggacga cagcatcgac aacaaagttt tgacccgtag cgacaagaat
     7441 cgcggtaaaa gcgataatgt tccgagcgaa gaggtcgtta agaagatgaa aaactattgg
     7501 cgtcagttgc tgaatgcaaa actgattacc caacgcaaat tcgacaattt gactaaagcg
     7561 gagcgtggtg gtctgagcga gctggacaag gcgggtttca ttaagcgtca actggttgaa
     7621 actcgtcaga tcacgaagca cgtggcgcag
//
