LOCUS       sequence-349457-        7779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human FLAG-tagged JAK1 Gly590Arg mutant expression (N-term. tag) 
            (CMV prom.).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7779)
  AUTHORS   Coelho MA, Cooper S, Strauss ME, Karakoc E, Bhosle S, Goncalves E, 
            Picco G, Burgold T, Cattaneo CM, Veninga V, Consonni S, Dincer C, 
            Vieira SF, Gibson F, Barthorpe S, Hardy C, Rein J, Thomas M, Marioni
            J, Voest EE, Bassett A, Garnett MJ
  TITLE     Base editing screens map mutations affecting interferon-gamma 
            signaling in cancer.
  JOURNAL   Cancer Cell. 2023 Feb 13;41(2):288-303.e6. doi: 
            10.1016/j.ccell.2022.12.009. Epub 2023 Jan 19.
  PUBMED    36669486
REFERENCE   2  (bases 1 to 7779)
  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: 
            "10.1016/j.ccell.2022.12.009"; journalName: "Cancer Cell"; date: 
            "2023-02-13- 13"; volume: "41"; issue: "2"; pages: "288-303.e6"
COMMENT     Sequence Label: FLAG-JAK1Gly590Arg
FEATURES             Location/Qualifiers
     source          1..7779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1253..1318
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4820..4837)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4826..5033
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5042..5182
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5042..5171
                     /label=AAV2 ITR
     rep_origin      5257..5712
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5344..5363)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5554..5575
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5729..5748)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5848..5870
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5908..5926)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5994..6098
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6099..6167,6168..6959)
                     /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     complement(6317..6336)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7130..7718
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7619..7638
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccrggg gggggggggg gggggggggg gggggggggg gsgrggsggr
      901 grggkgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa
     1261 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagtc
     1321 tggtggttct cagtatctaa atataaaaga ggactgcaat gccatggctt tctgtgctaa
     1381 aatgaggagc tccaagaaga ctgaggtgaa cctggaggcc cctgagccag gggtggaagt
     1441 gatcttctat ctgtcggaca gggagcccct ccggctgggc agtggagagt acacagcaga
     1501 ggaactgtgc atcagggctg cacaggcatg ccgtatctct cctctttgtc acaacctctt
     1561 tgccctgtat gacgagaaca ccaagctctg gtatgctcca aatcgcacca tcaccgttga
     1621 tgacaagatg tccctccggc tccactaccg gatgaggttc tatttcacca attggcatgg
     1681 aaccaacgac aatgagcagt cagtgtggcg tcattctcca aagaagcaga aaaatggcta
     1741 cgagaaaaaa aagattccag atgcaacccc tctccttgat gccagctcac tggagtatct
     1801 gtttgctcag ggacagtatg atttggtgaa atgcctggct cctattcgag accccaagac
     1861 cgagcaggat ggacatgata ttgagaacga gtgtctaggg atggctgtcc tggccatctc
     1921 acactatgcc atgatgaaga agatgcagtt gccagaactg cccaaggaca tcagctacaa
     1981 gcgatatatt ccagaaacat tgaataagtc catcagacag aggaaccttc tcaccaggat
     2041 gcggataaat aatgttttca aggatttcct aaaggaattt aacaacaaga ccatttgtga
     2101 cagcagcgtg tccacgcatg acctgaaggt gaaatacttg gctaccttgg aaactttgac
     2161 aaaacattac ggtgctgaaa tatttgagac ttccatgtta ctgatttcat cagaaaatga
     2221 gatgaattgg tttcattcga atgacggtgg aaacgttctc tactacgaag tgatggtgac
     2281 tgggaatctt ggaatccagt ggaggcataa accaaatgtt gtttctgttg aaaaggaaaa
     2341 aaataaactg aagcggaaaa aactggaaaa taaacacaag aaggatgagg agaaaaacaa
     2401 gatccgggaa gagtggaaca atttttctta cttccctgaa atcactcaca ttgtaataaa
     2461 ggagtctgtg gtcagcatta acaagcagga caacaagaaa atggaactga agctctcttc
     2521 ccacgaggag gccttgtcct ttgtgtccct ggtagatggc tacttccggc tcacagcaga
     2581 tgcccatcat tacctctgca ccgacgtggc ccccccgttg atcgtccaca acatacagaa
     2641 tggctgtcat ggtccaatct gtacagaata cgccatcaat aaattgcggc aagaaggaag
     2701 cgaggagggg atgtacgtgc tgaggtggag ctgcaccgac tttgacaaca tcctcatgac
     2761 cgtcacctgc tttgagaagt ctgagcaggt gcagggtgcc cagaagcagt tcaagaactt
     2821 tcagatcgag gtgcagaagg gccgctacag tctgcacggt tcggaccgca gcttccccag
     2881 cttgggagac ctcatgagcc acctcaagaa gcagatcctg cgcacggata acatcagctt
     2941 catgctaaaa cgctgctgcc agcccaagcc ccgagaaatc tccaacctgc tggtggctac
     3001 taagaaagcc caggagtggc agcccgtcta ccccatgagc cagctgagtt tcgatcggat
     3061 cctcaagaag gatctggtgc agggcgagca ccttaggaga ggcacgagaa cacacatcta
     3121 ttctgggacc ctgatggatt acaaggatga cgaaggaact tctgaagaga agaagataaa
     3181 agtgatcctc aaagtcttag accccagcca cagggatatt tccctggcct tcttcgaggc
     3241 agccagcatg atgagacagg tctcccacaa acacatcgtg tacctctatg gcgtctgtgt
     3301 ccgcgacgtg gagaatatca tggtggaaga gtttgtggaa gggggtcctc tggatctctt
     3361 catgcaccgg aaaagcgatg tccttaccac accatggaaa ttcaaagttg ccaaacagct
     3421 ggccagtgcc ctgagctact tggaggataa agacctggtc catggaaatg tgtgtactaa
     3481 aaacctcctc ctggcccgtg agggcatcga cagtgagtgt ggcccattca tcaagctcag
     3541 tgaccccggc atccccatta cggtgctgtc taggcaagaa tgcattgaac gaatcccatg
     3601 gattgctcct gagtgtgttg aggactccaa gaacctgagt gtggctgctg acaagtggag
     3661 ctttggaacc acgctctggg aaatctgcta caatggcgag atccccttga aagacaagac
     3721 gctgattgag aaagagagat tctatgaaag ccggtgcagg ccagtgacac catcatgtaa
     3781 ggagctggct gacctcatga cccgctgcat gaactatgac cccaatcaga ggcctttctt
     3841 ccgagccatc atgagagaca ttaataagct tgaagagcag aatccagata ttgtttcaga
     3901 aaaaaaacca gcaactgaag tggaccccac acattttgaa aagcgcttcc taaagaggat
     3961 ccgtgacttg ggagagggcc actttgggaa ggttgagctc tgcaggtatg accccgaagg
     4021 ggacaataca ggggagcagg tggctgttaa atctctgaag cctgagagtg gaggtaacca
     4081 catagctgat ctgaaaaagg aaatcgagat cttaaggaac ctctatcatg agaacattgt
     4141 gaagtacaaa ggaatctgca cagaagacgg aggaaatggt attaagctca tcatggaatt
     4201 tctgccttcg ggaagcctta aggaatatct tccaaagaat aagaacaaaa taaacctcaa
     4261 acagcagcta aaatatgccg ttcagatttg taaggggatg gactatttgg gttctcggca
     4321 atacgttcac cgggacttgg cagcaagaaa tgtccttgtt gagagtgaac accaagtgaa
     4381 aattggagac ttcggtttaa ccaaagcaat tgaaaccgat aaggagtatt acaccgtcaa
     4441 ggatgaccgg gacagccctg tgttttggta tgctccagaa tgtttaatgc aatctaaatt
     4501 ttatattgcc tctgacgtct ggtcttttgg agtcactctg catgagctgc tgacttactg
     4561 tgattcagat tctagtccca tggctttgtt cctgaaaatg ataggcccaa cccatggcca
     4621 gatgacagtc acaagacttg tgaatacgtt aaaagaagga aaacgcctgc cgtgcccacc
     4681 taactgtcca gatgaggttt atcaacttat gaggaaatgc tgggaattcc aaccatccaa
     4741 tcggacaagc tttcagaacc ttattgaagg atttgaagca cttttaaaat aagaattcta
     4801 actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     4861 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     4921 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4981 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
     5041 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     5101 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     5161 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5221 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5281 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5341 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5401 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5461 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5521 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5581 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     5641 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5701 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5761 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5821 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5881 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5941 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     6001 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6061 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6121 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6181 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6241 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6301 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6361 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6421 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6481 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6541 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6601 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6661 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6721 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6781 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     6841 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6901 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6961 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7021 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7081 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7141 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7201 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7261 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7321 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7381 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7441 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7501 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7561 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7621 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7681 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7741 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
