LOCUS       sequence-399083-        7659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for bacterial purification of codon optimized 
            evoFERNY-Cas9NG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7659)
  AUTHORS   Martin-Rufino JD, Castano N, Pang M, Grody EI, Joubran S, Caulier A,
            Wahlster L, Li T, Qiu X, Riera-Escandell AM, Newby GA, Al'Khafaji A,
            Chaudhary S, Black S, Weng C, Munson G, Liu DR, Wlodarski MW, Sims 
            K, Oakley JH, Fasano RM, Xavier RJ, Lander ES, Klein DE, Sankaran VG
  TITLE     Massively parallel base editing to map variant effects in human 
            hematopoiesis.
  JOURNAL   Cell. 2023 Apr 27:S0092-8674(23)00332-X. doi: 
            10.1016/j.cell.2023.03.035.
  PUBMED    37137305
REFERENCE   2  (bases 1 to 7659)
  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: "Cell. 2023 
            Apr 27:S0092-8674(23)00332-X. doi: 10.1016/j.cell.2023.03.035."
COMMENT     Sequence Label: evoFERNY-Cas9NG
FEATURES             Location/Qualifiers
     source          1..7659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     137..156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     353..370
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(501..528)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(552..1361)
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1275..1294
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1362..1452)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(1461..1491)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        1556..1674
                     /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             1709..1732
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1766..1786
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6518..6766
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6797..7045
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             7091..7111
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7132..7150)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7146..7193
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      7291..7320
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      join(7350..7659,1..236)
                     /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.
                     "
ORIGIN
        1 acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg gaatgagaca
       61 aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca ggagagcgca
      121 cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
      181 actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta tggaaaaacg
      241 gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt ccaggaaatc
      301 tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc gtagcgagtc
      361 agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca ccggtgcagc
      421 cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc aacatagtaa
      481 gccagtatac actccgctag cgcagaaagg cccacccgaa ggtgagccag gtgattacat
      541 ttgggccctc attagaaaaa ctcatcgagc atcaaatgaa attgcaattt attcatatca
      601 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
      661 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
      721 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
      781 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
      841 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
      901 attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa
      961 acaggaatcg agtgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     1021 gaatcaggat attcttctaa tacctggaac gctgtttttc cggggatcgc agtggtgagt
     1081 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagtgg cataaattcc
     1141 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     1201 tgtttcagaa acaactctgg cgcatcgggc ttcccataca agcgatagat tgtcgcacct
     1261 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     1321 tttaatcgcg gcctcgacgt ttcccgttga atatggctca tagctcctga aaatctcgat
     1381 aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt
     1441 acgtgccgat caagaagacg gtcaaaagcc tccggtcgga ggcttttgac tttctgctat
     1501 ggaggtcagg tatgatttaa atggtcagta ttgagcgata tctagagaat tcgtccacca
     1561 caattcagca aattgtgaac atcatcacgt tcatctttcc ctggttgcca atggcccatt
     1621 ttcctgtcag taacgagaag gtcgcgaatt caggcgcttt ttagactggt cgtaatgaaa
     1681 ttctttttaa gaaggagata tacatatgca ccatcaccat catcatcatc acaaacgtac
     1741 cgcagatggt agcgaatttg aaagcccgaa aaaaaagcgt aaagttggtt caggcggctc
     1801 gggtagcttt gaacgcaatt acgacccgcg tgagctgcgt aaagaaacct acctgttgta
     1861 tgagattaaa tggggtaaga gcggtaagct gtggcgtcat tggtgccaga acaacagaac
     1921 ccaacacgca gaggtgtatt ttctggaaaa cattttcaac gcccgtcgct tcaatccgag
     1981 cacgcactgt agcatcacct ggtatctgag ctggagcccg tgcgcggaat gttctcagaa
     2041 aattgttgac ttcctgaaag agcatccgaa cgttaatctg gagatttacg tcgcccgctt
     2101 atactacccg gagaacgaac gcaatcgtca gggtctgcgc gacctggtga atagcggcgt
     2161 tacgattcgc attatggatc tgccggatta taattactgc tggaaaacgt ttgttagcga
     2221 ccaaggtggt gacgaagatt actggccggg tcacttcgca ccttggatca aacagtactc
     2281 cctgaagctg tctggcggtt cgtctggtgg tagcagcggc agcgaaacgc cgggtaccag
     2341 cgagagcgca accccggaaa gctccggtgg cagctctggc ggctctgaca aaaagtattc
     2401 gattggcctg gcgatcggca cgaacagcgt cggttgggct gttattaccg acgagtataa
     2461 agtgccgagc aagaagttta aagtcttggg taacaccgat cgccatagca tcaaaaagaa
     2521 tctgattggt gccctgctgt ttgatagcgg cgaaacggca gaagcgaccc gtctgaaacg
     2581 taccgcacgt cgtcgctaca ctcgtcgtaa aaaccgcatt tgctatttgc aagagatctt
     2641 cagcaatgaa atggccaaag ttgacgacag cttctttcac cgtctggagg agagctttct
     2701 ggttgaagag gataagaaac acgagcgtca cccgatcttt ggtaatatcg ttgacgaggt
     2761 cgcgtatcac gagaagtacc cgacgatcta ccacttgcgt aagaaactgg tggacagcac
     2821 cgataaagcc gatctgcgtc tgatttactt ggcgttggcg cacatgatca agttccgcgg
     2881 tcatttcctg atcgagggtg acctgaaccc agataattcc gacgtcgaca agctgttcat
     2941 ccagctggtt cagacctaca accaattgtt tgaagaaaac ccgattaatg catctggtgt
     3001 cgacgcgaag gcgattctga gcgcgcgtct gagcaagtct cgtcgtctgg agaatctgat
     3061 cgcgcaattg ccgggtgaga agaaaaatgg tttgtttggc aacttgatcg cgctgagcct
     3121 gggtctgacc ccgaacttta agagcaattt cgacctggca gaagatgcga aactgcagct
     3181 gagcaaagac acctatgatg acgacctgga taatctcctg gcacaaattg gtgaccagta
     3241 tgcggacctg ttcctggctg cgaaaaatct gtcggacgcc attctgctgt cggacattct
     3301 gcgcgttaac actgagatca cgaaagcacc gctgtctgcg agcatgatta aacgttatga
     3361 cgagcaccat caagacctga ccctgctgaa ggccctggtc cgtcagcaac tgccggagaa
     3421 gtataaagaa atttttttcg atcagagcaa gaacggttac gcaggctata tcgatggcgg
     3481 tgcgagccaa gaagagtttt acaaattcat taaaccgatt ttggagaaga tggacggtac
     3541 cgaggagttg ctggtgaaac tgaatcgcga agatctgctg cgcaaacaac gtacgttcga
     3601 taacggtagc atcccacacc agattcacct gggtgagctg cacgcgattc tgcgtcgtca
     3661 ggaagatttc tacccattcc tgaaagataa tcgtgaaaag attgagaaaa tcttgacgtt
     3721 ccgcattccg tactacgtcg gtccgctggc acgtggcaac agccgtttcg cttggatgac
     3781 gcgcaagagc gaggaaacta tcacgccgtg gaactttgag gaagtcgtcg acaaaggtgc
     3841 gagcgcgcaa agctttattg agcgcatgac caactttgac aagaatctgc cgaacgagaa
     3901 agtcttgcca aagcacagcc tgctgtacga gtatttcacg gtgtacaatg aactgaccaa
     3961 agttaagtat gtcaccgagg gtatgcgtaa accagcgttc ctgtccggtg aacagaagaa
     4021 agcaattgtg gatctgttgt ttaagacgaa ccgtaaagtc acggtgaagc agttgaaaga
     4081 ggattacttc aaaaagatcg aatgctttga cagcgtggaa attagcggtg ttgaggaccg
     4141 tttcaacgcc agcttgggca cctaccatga tctgctgaaa atcattaagg acaaggactt
     4201 cctggataac gaagaaaacg aggatattct ggaggatatt gtcctgacgc tgaccctctt
     4261 cgaagatcgc gaaatgattg aagagagact gaaaacttac gcccacttgt ttgacgataa
     4321 agtcatgaaa caactgaagc gccgtcgtta caccggttgg ggtcgtctga gccgtaagct
     4381 gatcaacggt atccgtgata agcaatctgg caaaacgatc ctggactttc tgaaaagcga
     4441 tggtttcgcg aaccgcaact tcatgcagct tattcacgac gattctctga cctttaagga
     4501 ggacatccaa aaagcccagg ttagcggtca gggcgattcc ctgcatgagc atattgctaa
     4561 cttagcgggt agcccggcaa tcaaaaaggg cattctgcaa accgttaagg ttgtggacga
     4621 gttggttaag gtcatgggcc gtcataagcc ggagaatatc gttattgaga tggcgcgcga
     4681 gaatcagacc acgcagaaag gccaaaagaa tagccgtgaa cgtatgaaac gtattgaaga
     4741 gggtatcaaa gagctgggca gccaaatctt gaaggaacat ccggtggaga acacgcagct
     4801 gcagaatgaa aagctgtacc tgtactatct gcaaaacggt cgtgatatgt acgttgacca
     4861 agagctggac atcaaccgcc tgagcgatta tgatgttgat cacattgtgc cgcaatcttt
     4921 tctgaaggac gacagcatcg acaacaaagt tttgacccgt agcgacaaga atcgcggtaa
     4981 aagcgataat gttccgagcg aagaggtcgt taagaagatg aaaaactatt ggcgtcagtt
     5041 gctgaatgca aaactgatta cccaacgcaa attcgacaat ttgactaaag cggagcgtgg
     5101 tggtctgagc gagctggaca aggcgggttt cattaagcgt caactggttg aaactcgtca
     5161 gatcacgaag cacgtggcgc agattctgga cagccgcatg aatactaagt acgatgaaaa
     5221 tgataaactc attcgtgagg tcaaggtcat taccctgaag tctaagctcg tcagcgattt
     5281 tcgtaaagac ttccagttct ataaggtgcg cgaaatcaac aattaccacc acgcccatga
     5341 tgcatatctg aacgccgtgg tgggtacggc tctgatcaag aaatatccga aactggagtc
     5401 cgagtttgtc tatggtgact acaaagtgta tgacgtccgc aaaatgatcg ccaaatcgga
     5461 acaagagatt ggcaaggcaa ccgcgaagta ctttttctat tccaacatta tgaacttttt
     5521 caagaccgag atcaccctgg caaacggtga gatccgtaaa cgtccgctga tcgaaactaa
     5581 cggtgaaacc ggtgaaatcg tgtgggacaa aggtcgtgac tttgcaaccg tgcgtaaagt
     5641 gctgtcgatg ccacaggtca acatcgttaa gaaaaccgaa gttcaaactg gtggctttag
     5701 caaggagagc attcgtccta agcgcaacag cgacaagctg attgcacgta agaaggactg
     5761 ggacccgaag aagtacggcg gctttgtgtc cccgacggtt gcgtatagcg ttctcgttgt
     5821 ggcgaaagtc gagaaaggca agagcaaaaa gctgaaaagc gttaaagaat tgctgggtat
     5881 cacgattatg gagcgtagca gcttcgaaaa gaatcctatt gatttcttgg aagcaaaagg
     5941 ttacaaggag gtgaagaaag acttgatcat caaactgccg aagtattccc tgttcgagct
     6001 ggaaaacggc cgcaaacgta tgctggccag cgctcgcttt ctgcaaaagg gcaacgaact
     6061 ggctctgccg agcaaatatg tgaatttcct gtacctggcc agccactacg agaaactgaa
     6121 gggctccccg gaagataacg agcagaagca gctgttcgtt gagcagcaca agcattacct
     6181 ggacgagatt atcgagcaga tcagcgagtt tagcaaacgc gtgattctgg cggatgcgaa
     6241 tttggataaa gttctgtcgg cctataataa acaccgcgac aaaccgattc gcgagcaggc
     6301 ggagaacatt atccacttgt tcaccctgac caatctgggt gctccgcgtg ctttcaaata
     6361 ctttgatacc acgattgatc gcaaagtgta tcgttccacc aaagaggtac tggacgcgac
     6421 cctgatccac cagagcatca ccggcttgta cgaaacgcgc atcgatctgt ctcaactggg
     6481 cggtgacagc ggtggttcgg gtggtagcgg tggcagcacc aacctgagcg acattattga
     6541 aaaagaaacc ggcaaacaac tggtcatcca agaaagcatt ctgatgttgc cggaggaagt
     6601 tgaagaggtg attggcaata aaccggagag cgatatcctg gtgcataccg cgtacgacga
     6661 aagcactgat gagaatgtga tgttgctgac ctccgatgca ccggagtaca aaccgtgggc
     6721 gttggtgatc caagatagca atggtgaaaa caagattaag atgctgtccg gtggttccgg
     6781 tggctctggt ggttccacca acttgtccga catcattgag aaagaaaccg gtaagcagct
     6841 ggttattcaa gaatctattc tgatgctgcc ggaagaggtt gaagaagtca tcggcaataa
     6901 gcctgagagc gacattttgg ttcataccgc ttacgatgag agcacggatg aaaatgtaat
     6961 gctgctgacg agcgacgctc cggagtacaa gccgtgggca ctggtgattc aagatagcaa
     7021 cggcgagaat aagatcaaaa tgctgagcgg tggtagcaaa cgcacagccg atggatcaga
     7081 atttgaatct ccgaaaaaga aacgcaaggt gtaaggttag agcggccgcc accgctgagc
     7141 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     7201 gaggaactat atccgggtaa cgaattcaag cttgatatca ttcaggacga gcctcagact
     7261 ccagcgtaac tggactgcaa tcaactcact ggctcacctt cacgggtggg cctttcttcg
     7321 gtagaagtct tcttaataag atgatcttct tgagatcgtt ttggtctgcg cgtaatctct
     7381 tgctctgaaa acgaaaaaac cgccttgcag ggcggttttt cgaaggttct ctgagctacc
     7441 aactctttga accgaggtaa ctggcttgga ggagcgcagt caccaaaact tgtcctttca
     7501 gtttagcctt aaccggcgca tgacttcaag actaactcct ctaaatcaat taccagtggc
     7561 tgctgccagt ggtgcttttg catgtctttc cgggttggac tcaagacgat agttaccgga
     7621 taaggcgcag cggtcggact gaacgggggg ttcgtgcat
//
