LOCUS       sequence-434423-        8315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal BirA*-tagged human wild type TLK1 variant 5 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  JOURNAL   Unpublished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            "Unpublished"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-WT
FEATURES             Location/Qualifiers
     source          1..8315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..86
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        161..265
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(266..334,335..1126)
                     /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(484..503)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1297..1885
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1786..1805
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2039..2056
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2173..2194
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2209..2214,2215..2232,2233..2239)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2247..2263
                     /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)."
     primer_bind     2252..2274
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2271..2287
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2271..2287
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2336..2457
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2347..2366)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2401..2420
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2587..3609)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(2587..3609)
                     /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
                     "
     protein_bind    complement(3617..3664)
                     /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)."
     primer_bind     3796..3815
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3948..4172
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4161..4178
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(6415..7023,7024..7026,7027..7377))
                     /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"
     CDS             complement(7429..7452)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(7552..7576)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    7586..7604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7607..7625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(7627..7830)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(7656..7676)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        7831..8210
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ttttgctcgc gcactactca gcgacctcca acacacaagc agggagcaga tactggctta
       61 actatgcggc atcagagcag attgtactga gagtgcacca taggggatcg ggagatctcc
      121 cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
      181 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
      241 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
      301 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
      361 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
      421 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
      481 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
      541 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
      601 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
      661 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
      721 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
      781 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
      841 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
      901 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
      961 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     1021 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     1081 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     1141 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     1201 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     1261 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     1321 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     1381 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     1441 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     1501 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     1561 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     1621 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     1681 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     1741 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     1801 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     1861 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     1921 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     1981 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     2041 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     2101 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     2161 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     2221 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     2281 ctatgaccat gattacgcca agctctagct agaggtcgac ggtatacaga catgataaga
     2341 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     2401 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttggg
     2461 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg
     2521 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtag
     2581 tacgtgctat tcctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag
     2641 tacttctaca cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc
     2701 gacagtcccg gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc
     2761 atcgaaattg ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata
     2821 cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg
     2881 ctgctccata caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga
     2941 atccccgaac atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag
     3001 gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc
     3061 ccaaagcatc agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac
     3121 agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt
     3181 gtattgaccg attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc
     3241 cgcagcgatc gcatccatgg cctccgcgac cggctgcaga acagcgggca gttcggtttc
     3301 aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg tcaggctctc
     3361 gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg
     3421 ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc
     3481 acgccctcct acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag
     3541 gtcggagacg ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc
     3601 aggctttttg gccaaggaag ttcctatact ttctagagaa taggaacttc ggaataggaa
     3661 cttctaggta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3721 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3781 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3841 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3901 gtggggatac cccctagagc cccagctggt tctttccgcc tcagaagcca tagagcccac
     3961 cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac
     4021 cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta
     4081 ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca
     4141 aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcggg tttaaacggg
     4201 ccctctagac tcgagttagg cggccgctca gtaagtaatt atgcttgaag aagggggtgt
     4261 aggggatgct gtcagcccag ccatgtgtag gtttcctgaa gaatttgatc ttctcatgtg
     4321 tgggagaagg tatgggtcat ttgccagctg gtgcacatca aatcgatctt cttttcgata
     4381 tgccaaacag cgtcttataa atgccttggc ttcactgctt acaaccggtt ttacagggaa
     4441 ctggacttct gtggctttta atattgtatt ttcttgaaga atgtcttgtt gagattgatt
     4501 gtgaccaaat ggctttctac cataaagaca ctgaaagaag atgactccaa ccgaccatac
     4561 atcaaccttg ttggaaatct ttggtggctc ttttccaact acaaaacact caggaggtaa
     4621 ataccagtaa gtgcctgccc cctgggaagt tagatccatt ccatctacac catagctatc
     4681 atcatccata atcttggaca gaccaaaatc agtgattttg atttcaccac atgctgttcc
     4741 atctaccagt aggatgtttc ctggcttaag atcataatgt ataatagggg gtttgatctc
     4801 attgagatat cttagtgcat ttacaatctg cattacaata gaccgagctt ctttctctga
     4861 cattaacttg tgttgcttca gatagaaatc caagtcattg ccttcacagt attctaacac
     4921 tgtacaaaac gtatctgtat ccaaggagaa ataatcatag agtttaacta ttctggggtg
     4981 atccagttct ttgtgtattc tatactctct gcaggcatgt ttgtggtagt tttctttctt
     5041 ctcatctctc cagcttttat taagctgatg tatcttcaca gcagcatatc tttgttcata
     5101 aaggtcaaaa gccttataca cttcactaaa gccacctcta ccaagcagat gaagtaataa
     5161 atatctttca tttaatgttg ggtgatcttt gaactgtgaa ttatcttcat tgtttattct
     5221 tttcagctca cgtatgtgaa gatttctgac tctttccaaa cgttcaagtt ctgcctggat
     5281 ttctgcctct tcctttttga gatgtcctag tctaagtttg aaaatttctt cctgttcatg
     5341 atattctgcc aaagtcaaca gttgtggtaa atttggtcta acaaagggat cattctctgc
     5401 tccattgact gctttgtttt tcctttgttt tggttcagaa ttggtagagg gtgcctgaga
     5461 attattagct gtgggaggtt tgcgtttggc tagaagtttc ctttgccttt caatatcttc
     5521 cctttgctga ttcacccatt cttgttgctt cacaagattc tgaaatgcaa aaccatctgt
     5581 ccattgttca gtaaatgaag cgccatgtct aactgttgta aagtgcccaa ggcgtaatcg
     5641 atcttgcata ctcttctctc tgcttgacag cttttcttgt gtactctttt caataagaag
     5701 tttcttgctc attgatatgc acttatttaa tcgttctttg tatttttcaa gtaatttttg
     5761 ttgttcatct atttgccgtc tgagatcaca gttagccctg agcaaatcat ctatacgtcc
     5821 ctccttcttt tccaggtcct ggattttatt actttctaat gctgctaatt tcagcattgt
     5881 gagatcagtc tgaataattt taaaggataa ttgctttggt tgtacaatag ggtggtcccc
     5941 aaatgctaat gcagtaggag aagggctatt cggtcgaaca gatgaggaag gagtggaatg
     6001 tgaatgtgaa ttttgaggag aacggattgc aggaggtatg cctcttactg gacttgagcc
     6061 atttccaccc tggtattcaa aatagtcgct aattttgtgg ccacgtcccc caatactttt
     6121 tccctgacta ctttcattct ggttttctgc ttttctcttt cttcccctgg atgattccga
     6181 ttgtttcttc tccggtgtct ctgattcttt gtcacttaaa gatcccaagc ttccaaaact
     6241 gtgattagag ctttcgttat ttgttgaggc tttagctcca acactgcaac tgcccgtact
     6301 tccagtgctc ccacttgcaa ctccagtaaa tctagcttcc aataactctt gccttcttgg
     6361 atccagacta tgaagctcat ccataggcgc gccgccgccg gggccagatc cccccttctc
     6421 ggcgctccgc aggctgatct cgccgcccat ccagggcttg atgatgccgt cctgctccag
     6481 cagcagggcg ccctgcttgt cgatgccccg gctgatgccg aagatctcct tgtcgccgat
     6541 gatcagcttc acgggccggt tgatgaagtt gtccagcttc tcccaccggc tcaggtaggg
     6601 ggccaggccc tcctgctcga acagctccag ggcggcccgc agctcccgga tcagcatggc
     6661 ggccagggtg ttccggtcca ggttgatgcc ggcctcctgc agggtgatcc agccctggtt
     6721 caccacgctc tcctccaccc gccgcatggc catgttgatg ccggcgccga tcacgatctg
     6781 ggcggcgtcg ccggtcttgc cggtcagctc caccaggatg ccggccagct tccggtcctg
     6841 caggtacagg tcgttgggcc acttcacccg caccttgtcg gcgcccagct tccgcagcac
     6901 ctcggccatc acgatgccga tcaccaggct caggccgatg gcggcggcgg ggccctgctc
     6961 cagccgccag aacatgctca ggtacaggtt ggcgccgaag gggctgaacc acttccggcc
     7021 ccggccgccc cggccggcct gctggtactc ggcgatgcag gcgtcgccgc tcttcagctc
     7081 gccgatccgg tccagcaggt actggttggt gctgtcgatc acgggcagca cggccacgct
     7141 gccgccgtcc agctggccca ggatctgctt ggcgttcagc agctggatgg gctcgggcag
     7201 gctgtagccc ttgccgggca cggtgaacac gtccacgccc cagtcccgca gggtctggat
     7261 gtgcttgttg atggcggccc ggctcatgcc cagggtctcg cccagctgct cgccgctgtg
     7321 gaactcgccg ttggccagca gggcgatcag cttcaggggc acggtgttgt ccttcatggt
     7381 ggcgctagct ggatccttaa ttaatggcgc accgtttaaa ctggcggctt tatcgtcatc
     7441 gtctttgtag tccatggtac caagcttaag tttaaacgct agagtccgga ggctggatcg
     7501 gtcccggtgt cttctatgga ggtcaaaaca gcgtggatgg cgtctccagg cgatctgacg
     7561 gttcactaaa cgagctcgtc gacgatctct atcactgata gggagatctc tatcactgat
     7621 agggagagct ctgcttatat agacctccca ccgtacacgc ctaccgccca tttgcgtcaa
     7681 tggggcggag ttgttacgac attttggaaa gtcccgttga ttttggtgcc aaaacaaact
     7741 cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc gctatccacg
     7801 cccattgatg tactgccaaa accgcatcac catggtaata gcgatgacta atacgtagat
     7861 gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca
     7921 tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc
     7981 caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg
     8041 cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca
     8101 gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac
     8161 taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc aacgcgtata
     8221 tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca
     8281 attgtcggtc aagccttgcc ttgttgtagc ttaaa
//
