LOCUS       sequence-434421-        8315 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses N-terminal BirA*-tagged human TLK1 variant 5 mutant Q479E 
            for BioID experiments..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8315)
  TITLE     Identification of de novo mutations in TLK1 and MDM1 in a patient 
            with a neurodevelopmental disorder and immunodeficiency
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8315)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pcDNA5/FRT/TO-N-FLAG-hBirA*-TLK1-Q479E
FEATURES             Location/Qualifiers
     source          1..8315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(110..127)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    116..340
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(473..492)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    624..671
                     /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             679..1701
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             679..1701
                     /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    1831..1952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1868..1887)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1922..1941
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2001..2017)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2001..2017)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2014..2036)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2025..2041
                     /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(2049..2055,2056..2073,2074..2079))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2094..2115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2232..2249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2403..2991)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2483..2502)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3162..3953,3954..4022))
                     /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     3785..3804
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4023..4127)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4202..4221)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4393..4772
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4773..4976
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4927..4947
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4978..4996
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4999..5017
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5027..5051
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5151..5174
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(5226..5576,5577..5579,5580..6188)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
ORIGIN
        1 cacatggctg ggctgacagc atcccctaca cccccttctt caagcataat tacttactga
       61 gcggccgcct aactcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
      121 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
      181 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
      241 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
      301 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
      361 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
      421 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
      481 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      541 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      601 tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt
      661 ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
      721 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
      781 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
      841 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
      901 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
      961 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     1021 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     1081 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     1141 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     1201 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     1261 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     1321 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     1381 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     1441 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     1501 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     1561 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     1621 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     1681 cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat
     1741 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     1801 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     1861 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     1921 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     1981 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     2041 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     2101 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2161 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2221 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2281 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2341 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2401 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2461 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2521 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2581 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2641 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2701 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2761 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2821 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2881 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2941 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3001 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3061 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3121 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3181 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3241 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3301 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3361 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3421 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3481 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3541 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3601 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3661 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3721 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3781 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3841 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3901 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3961 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4021 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4081 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4141 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     4201 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     4261 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     4321 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     4381 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     4441 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     4501 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     4561 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     4621 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     4681 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     4741 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     4801 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     4861 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     4921 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc
     4981 ctatcagtga tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga
     5041 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg
     5101 accgatccag cctccggact ctagcgttta aacttaagct tggtaccatg gactacaaag
     5161 acgatgacga taaagccgcc agtttaaacg gtgcgccatt aattaaggat ccagctagcg
     5221 ccaccatgaa ggacaacacc gtgcccctga agctgatcgc cctgctggcc aacggcgagt
     5281 tccacagcgg cgagcagctg ggcgagaccc tgggcatgag ccgggccgcc atcaacaagc
     5341 acatccagac cctgcgggac tggggcgtgg acgtgttcac cgtgcccggc aagggctaca
     5401 gcctgcccga gcccatccag ctgctgaacg ccaagcagat cctgggccag ctggacggcg
     5461 gcagcgtggc cgtgctgccc gtgatcgaca gcaccaacca gtacctgctg gaccggatcg
     5521 gcgagctgaa gagcggcgac gcctgcatcg ccgagtacca gcaggccggc cggggcggcc
     5581 ggggccggaa gtggttcagc cccttcggcg ccaacctgta cctgagcatg ttctggcggc
     5641 tggagcaggg ccccgccgcc gccatcggcc tgagcctggt gatcggcatc gtgatggccg
     5701 aggtgctgcg gaagctgggc gccgacaagg tgcgggtgaa gtggcccaac gacctgtacc
     5761 tgcaggaccg gaagctggcc ggcatcctgg tggagctgac cggcaagacc ggcgacgccg
     5821 cccagatcgt gatcggcgcc ggcatcaaca tggccatgcg gcgggtggag gagagcgtgg
     5881 tgaaccaggg ctggatcacc ctgcaggagg ccggcatcaa cctggaccgg aacaccctgg
     5941 ccgccatgct gatccgggag ctgcgggccg ccctggagct gttcgagcag gagggcctgg
     6001 ccccctacct gagccggtgg gagaagctgg acaacttcat caaccggccc gtgaagctga
     6061 tcatcggcga caaggagatc ttcggcatca gccggggcat cgacaagcag ggcgccctgc
     6121 tgctggagca ggacggcatc atcaagccct ggatgggcgg cgagatcagc ctgcggagcg
     6181 ccgagaaggg gggatctggc cccggcggcg gcgcgcctat ggatgagctt catagtctgg
     6241 atccaagaag gcaagagtta ttggaagcta gatttactgg agttgcaagt gggagcactg
     6301 gaagtacggg cagttgcagt gttggagcta aagcctcaac aaataacgaa agctctaatc
     6361 acagttttgg aagcttggga tctttaagtg acaaagaatc agagacaccg gagaagaaac
     6421 aatcggaatc atccagggga agaaagagaa aagcagaaaa ccagaatgaa agtagtcagg
     6481 gaaaaagtat tgggggacgt ggccacaaaa ttagcgacta ttttgaatac cagggtggaa
     6541 atggctcaag tccagtaaga ggcatacctc ctgcaatccg ttctcctcaa aattcacatt
     6601 cacattccac tccttcctca tctgttcgac cgaatagccc ttctcctact gcattagcat
     6661 ttggggacca ccctattgta caaccaaagc aattatcctt taaaattatt cagactgatc
     6721 tcacaatgct gaaattagca gcattagaaa gtaataaaat ccaggacctg gaaaagaagg
     6781 agggacgtat agatgatttg ctcagggcta actgtgatct cagacggcaa atagatgaac
     6841 aacaaaaatt acttgaaaaa tacaaagaac gattaaataa gtgcatatca atgagcaaga
     6901 aacttcttat tgaaaagagt acacaagaaa agctgtcaag cagagagaag agtatgcaag
     6961 atcgattacg ccttgggcac tttacaacag ttagacatgg cgcttcattt actgaacaat
     7021 ggacagatgg ttttgcattt cagaatcttg tgaagcaaca agaatgggtg aatcagcaaa
     7081 gggaagatat tgaaaggcaa aggaaacttc tagccaaacg caaacctccc acagctaata
     7141 attctcaggc accctctacc aattctgaac caaaacaaag gaaaaacaaa gcagtcaatg
     7201 gagcagagaa tgatcccttt gttagaccaa atttaccaca actgttgact ttggcagaat
     7261 atcatgaaca ggaagaaatt ttcaaactta gactaggaca tctcaaaaag gaagaggcag
     7321 aaatccaggc agaacttgaa cgtttggaaa gagtcagaaa tcttcacata cgtgagctga
     7381 aaagaataaa caatgaagat aattcacagt tcaaagatca cccaacatta aatgaaagat
     7441 atttattact tcatctgctt ggtagaggtg gctttagtga agtgtataag gcttttgacc
     7501 tttatgaaga aagatatgct gctgtgaaga tacatcagct taataaaagc tggagagatg
     7561 agaagaaaga aaactaccac aaacatgcct gcagagagta tagaatacac aaagaactgg
     7621 atcaccccag aatagttaaa ctctatgatt atttctcctt ggatacagat acgttttgta
     7681 cagtgttaga atactgtgaa ggcaatgact tggatttcta tctgaagcaa cacaagttaa
     7741 tgtcagagaa agaagctcgg tctattgtaa tgcagattgt aaatgcacta agatatctca
     7801 atgagatcaa accccctatt atacattatg atcttaagcc aggaaacatc ctactggtag
     7861 atggaacagc atgtggtgaa atcaaaatca ctgattttgg tctgtccaag attatggatg
     7921 atgatagcta tggtgtagat ggaatggatc taacttccca gggggcaggc acttactggt
     7981 atttacctcc tgagtgtttt gtagttggaa aagagccacc aaagatttcc aacaaggttg
     8041 atgtatggtc ggttggagtc atcttctttc agtgtcttta tggtagaaag ccatttggtc
     8101 acaatcaatc tcaacaagac attcttcaag aaaatacaat attaaaagcc acagaagtcc
     8161 agttccctgt aaaaccggtt gtaagcagtg aagccaaggc atttataaga cgctgtttgg
     8221 catatcgaaa agaagatcga tttgatgtgc accagctggc aaatgaccca taccttctcc
     8281 cacacatgag aagatcaaat tcttcaggaa accta
//
