LOCUS       sequence-434422-        8315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal BirA*-tagged human TLK1 variant 5 mutant M566T 
            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  JOURNAL   Unpublished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 Dec 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            "Unpublished"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-M566T
FEATURES             Location/Qualifiers
     source          1..8315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        123..326
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     277..297
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    328..346
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    349..367
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     377..401
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             501..524
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(576..926,927..929,930..1538)
                     /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"
     primer_bind     complement(3775..3792)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3781..4005
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4138..4157)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4289..4336
                     /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             4344..5366
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4344..5366
                     /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    5496..5617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5533..5552)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5587..5606
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5666..5682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5666..5682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5679..5701)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5690..5706
                     /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(5714..5720,5721..5738,5739..5744))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5759..5780
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5897..5914)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6068..6656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6148..6167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6827..7618,7619..7687))
                     /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     7450..7469
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7688..7792)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7867..7886)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8058..8315,1..122)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
       61 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      121 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      181 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      241 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      301 ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc cctatcagtg
      361 atagagatcg tcgacgagct cgtttagtga accgtcagat cgcctggaga cgccatccac
      421 gctgttttga cctccataga agacaccggg accgatccag cctccggact ctagcgttta
      481 aacttaagct tggtaccatg gactacaaag acgatgacga taaagccgcc agtttaaacg
      541 gtgcgccatt aattaaggat ccagctagcg ccaccatgaa ggacaacacc gtgcccctga
      601 agctgatcgc cctgctggcc aacggcgagt tccacagcgg cgagcagctg ggcgagaccc
      661 tgggcatgag ccgggccgcc atcaacaagc acatccagac cctgcgggac tggggcgtgg
      721 acgtgttcac cgtgcccggc aagggctaca gcctgcccga gcccatccag ctgctgaacg
      781 ccaagcagat cctgggccag ctggacggcg gcagcgtggc cgtgctgccc gtgatcgaca
      841 gcaccaacca gtacctgctg gaccggatcg gcgagctgaa gagcggcgac gcctgcatcg
      901 ccgagtacca gcaggccggc cggggcggcc ggggccggaa gtggttcagc cccttcggcg
      961 ccaacctgta cctgagcatg ttctggcggc tggagcaggg ccccgccgcc gccatcggcc
     1021 tgagcctggt gatcggcatc gtgatggccg aggtgctgcg gaagctgggc gccgacaagg
     1081 tgcgggtgaa gtggcccaac gacctgtacc tgcaggaccg gaagctggcc ggcatcctgg
     1141 tggagctgac cggcaagacc ggcgacgccg cccagatcgt gatcggcgcc ggcatcaaca
     1201 tggccatgcg gcgggtggag gagagcgtgg tgaaccaggg ctggatcacc ctgcaggagg
     1261 ccggcatcaa cctggaccgg aacaccctgg ccgccatgct gatccgggag ctgcgggccg
     1321 ccctggagct gttcgagcag gagggcctgg ccccctacct gagccggtgg gagaagctgg
     1381 acaacttcat caaccggccc gtgaagctga tcatcggcga caaggagatc ttcggcatca
     1441 gccggggcat cgacaagcag ggcgccctgc tgctggagca ggacggcatc atcaagccct
     1501 ggatgggcgg cgagatcagc ctgcggagcg ccgagaaggg gggatctggc cccggcggcg
     1561 gcgcgcctat ggatgagctt catagtctgg atccaagaag gcaagagtta ttggaagcta
     1621 gatttactgg agttgcaagt gggagcactg gaagtacggg cagttgcagt gttggagcta
     1681 aagcctcaac aaataacgaa agctctaatc acagttttgg aagcttggga tctttaagtg
     1741 acaaagaatc agagacaccg gagaagaaac aatcggaatc atccagggga agaaagagaa
     1801 aagcagaaaa ccagaatgaa agtagtcagg gaaaaagtat tgggggacgt ggccacaaaa
     1861 ttagcgacta ttttgaatac cagggtggaa atggctcaag tccagtaaga ggcatacctc
     1921 ctgcaatccg ttctcctcaa aattcacatt cacattccac tccttcctca tctgttcgac
     1981 cgaatagccc ttctcctact gcattagcat ttggggacca ccctattgta caaccaaagc
     2041 aattatcctt taaaattatt cagactgatc tcacaatgct gaaattagca gcattagaaa
     2101 gtaataaaat ccaggacctg gaaaagaagg agggacgtat agatgatttg ctcagggcta
     2161 actgtgatct cagacggcaa atagatgaac aacaaaaatt acttgaaaaa tacaaagaac
     2221 gattaaataa gtgcatatca atgagcaaga aacttcttat tgaaaagagt acacaagaaa
     2281 agctgtcaag cagagagaag agtatgcaag atcgattacg ccttgggcac tttacaacag
     2341 ttagacatgg cgcttcattt actgaacaat ggacagatgg ttttgcattt cagaatcttg
     2401 tgaagcaaca agaatgggtg aatcagcaaa gggaagatat tgaaaggcaa aggaaacttc
     2461 tagccaaacg caaacctccc acagctaata attctcaggc accctctacc aattctgaac
     2521 caaaacaaag gaaaaacaaa gcagtcaatg gagcagagaa tgatcccttt gttagaccaa
     2581 atttaccaca actgttgact ttggcagaat atcatgaaca ggaagaaatt ttcaaactta
     2641 gactaggaca tctcaaaaag gaagaggcag aaatccaggc agaacttgaa cgtttggaaa
     2701 gagtcagaaa tcttcacata cgtgagctga aaagaataaa caatgaagat aattcacagt
     2761 tcaaagatca cccaacatta aatgaaagat atttattact tcatctgctt ggtagaggtg
     2821 gctttagtga agtgtataag gcttttgacc tttatgaaca aagatatgct gctgtgaaga
     2881 tacatcagct taataaaagc tggagagatg agaagaaaga aaactaccac aaacatgcct
     2941 gcagagagta tagaatacac aaagaactgg atcaccccag aatagttaaa ctctatgatt
     3001 atttctcctt ggatacagat acgttttgta cagtgttaga atactgtgaa ggcaatgact
     3061 tggatttcta tctgaagcaa cacaagttaa tgtcagagaa agaagctcgg tctattgtaa
     3121 cgcagattgt aaatgcacta agatatctca atgagatcaa accccctatt atacattatg
     3181 atcttaagcc aggaaacatc ctactggtag atggaacagc atgtggtgaa atcaaaatca
     3241 ctgattttgg tctgtccaag attatggatg atgatagcta tggtgtagat ggaatggatc
     3301 taacttccca gggggcaggc acttactggt atttacctcc tgagtgtttt gtagttggaa
     3361 aagagccacc aaagatttcc aacaaggttg atgtatggtc ggttggagtc atcttctttc
     3421 agtgtcttta tggtagaaag ccatttggtc acaatcaatc tcaacaagac attcttcaag
     3481 aaaatacaat attaaaagcc acagaagtcc agttccctgt aaaaccggtt gtaagcagtg
     3541 aagccaaggc atttataaga cgctgtttgg catatcgaaa agaagatcga tttgatgtgc
     3601 accagctggc aaatgaccca taccttctcc cacacatgag aagatcaaat tcttcaggaa
     3661 acctacacat ggctgggctg acagcatccc ctacaccccc ttcttcaagc ataattactt
     3721 actgagcggc cgcctaactc gagtctagag ggcccgttta aacccgctga tcagcctcga
     3781 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3841 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3901 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3961 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     4021 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     4081 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4141 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4201 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4261 ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag
     4321 aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     4381 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     4441 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     4501 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     4561 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     4621 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     4681 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     4741 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     4801 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     4861 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     4921 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     4981 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     5041 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     5101 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     5161 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     5221 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     5281 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     5341 gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct
     5401 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5461 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5521 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5581 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5641 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5701 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5761 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5821 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5881 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5941 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6001 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6061 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6121 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6181 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6241 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6301 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6361 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6421 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6481 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6541 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6601 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6661 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6721 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6781 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6841 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6901 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6961 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7021 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7081 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7141 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7201 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7261 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7321 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7381 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7441 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7501 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7561 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7621 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7681 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7741 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7801 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     7861 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     7921 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     7981 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     8041 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     8101 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     8161 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     8221 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     8281 ccacttggca gtacatcaag tgtatcatat gccaa
//
