LOCUS       sequence-203446-        6274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible gene expression of FlagHA-PRKRA sgRNA PAM mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6274)
  AUTHORS   Meyer C, Garzia A, Mazzola M, Gerstberger S, Molina H, Tuschl T
  TITLE     The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated 
            Stress Response and Cell Cycle Progression.
  JOURNAL   Mol Cell. 2018 Feb 15;69(4):622-635.e6. doi: 
            10.1016/j.molcel.2018.01.011.
  PUBMED    29429924
REFERENCE   2  (bases 1 to 6274)
  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.molcel.2018.01"; journalName: "Mol Cell"; date: 
            "2018-02-15- 15"; volume: "69"; issue: "4"; pages: "622-635.e6"
COMMENT     Sequence Label: pFRT_TO_FLAGHA_PRKRA_sgRNA_PAM_mutant
FEATURES             Location/Qualifiers
     source          1..6274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             759..782
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             783..809
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    825..849
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1794..1818)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1871..1912
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1992..2009)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1998..2222
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2355..2374)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2506..2553
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2561..3583
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2561..3583
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3713..3834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3750..3769)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3804..3823
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3883..3899)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3883..3899)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3896..3918)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3907..3923
                     /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)."
     promoter        complement(join(3931..3937,3938..3955,3956..3961))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3976..3997
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4114..4131)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4285..4873)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4365..4384)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5044..5835,5836..5904))
                     /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     5667..5686
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5905..6009)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6084..6103)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagcttg gtaccatgga ctacaaggac gacgatgaca
      781 agtaccctta tgacgtgccc gattacgctt tgaaccagat atcaacaagt ttgtacaaaa
      841 aagcaggctt catgtcccag agcaggcacc gcgccgaggc cccgccgctg gagcgcgagg
      901 acagtgggac cttcagtttg gggaagatga taacagctaa gccaggtaaa acaccgattc
      961 aggtattaca cgaatacggc atgaagacca agaacatccc agtttatgaa tgtgaaagat
     1021 ctgatgtgca aatacacgtg cccactttca ccttcagagt aaccgttggt gacataacct
     1081 gcacaggtga aggtacaagt aagaagctgg cgaaacatag agctgcagag gctgccataa
     1141 acattttgaa agccaatgca agtatttgct ttgcagttcc tgacccctta atgcctgacc
     1201 cttccaagca accaaagaac cagcttaatc ctattggttc attacaggaa ttggctattc
     1261 atcatggctg gagacttcct gaatataccc tttcccagga gggaggacct gctcataaga
     1321 gagaatatac tacaatttgc aggctagagt catttatgga aactggaaag ggggcatcaa
     1381 aaaagcaagc caaaaggaat gctgctgaga aatttcttgc caaatttagt aatatttctc
     1441 cagagaacca catttcttta acaaatgtag taggacattc tttaggatgt acttggcatt
     1501 ccttgaggaa ttctcctggt gaaaagatca acttactgaa aagaagcctc cttagtattc
     1561 caaatacaga ttacatccag ctgcttagtg aaattgccaa ggaacaaggt tttaatataa
     1621 catatttgga tatagatgaa ctgagcgcca atggacaata tcaatgtctt gctgaactgt
     1681 ccaccagccc catcacagtc tgtcatggct ccggtatctc ctgtggcaat gcacaaagtg
     1741 atgcagctca caatgctttg cagtatttaa agataatagc agaaagaaag taaacccagc
     1801 tttcttgtac aaagtggttg atatccagca cagtggcggc cgctcaagtc tagagggccc
     1861 gcggttcgaa ggtaagccta tccctaaccc tctcctcggt ctcgattcta cgcgtacctg
     1921 atccgcggcc gcatagataa ctgatccagt gtgcctcgag tctagagggc ccgtttaaac
     1981 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2041 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2101 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2161 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2221 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2281 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2341 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2401 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2461 cctcgacccc aaaaaacttg attagggtga tggttcacgt acctagaagt tcctattccg
     2521 aagttcctat tctctagaaa gtataggaac ttccttggcc aaaaagcctg aactcaccgc
     2581 gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct
     2641 ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct
     2701 gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc
     2761 atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac
     2821 ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact
     2881 gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag
     2941 ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg
     3001 tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga
     3061 caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg
     3121 ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa
     3181 tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga
     3241 ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta
     3301 cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg
     3361 cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg
     3421 ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca
     3481 aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag
     3541 tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt
     3601 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     3661 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     3721 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     3781 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     3841 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     3901 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3961 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4021 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4081 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4141 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4201 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4261 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4321 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4381 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4441 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4501 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4561 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4621 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4681 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4741 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4801 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4861 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4921 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4981 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5041 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5101 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5161 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5221 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5281 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5341 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5401 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5461 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5521 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5581 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5641 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5701 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5761 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5821 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5881 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5941 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6001 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     6061 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     6121 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     6181 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     6241 ctgcttcgcg atgtacgggc cagatatacg cgtt
//
