LOCUS       sequence-317454-        8249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Lck-TurboID in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8249)
  AUTHORS   Chua XY, Aballo T, Elnemer W, Tran M, Salomon A
  TITLE     Quantitative Interactomics of Lck-TurboID in Living Human T Cells 
            Unveils T Cell Receptor Stimulation-Induced Proximal Lck 
            Interactors.
  JOURNAL   J Proteome Res. 2020 Nov 13. doi: 10.1021/acs.jproteome.0c00616.
  PUBMED    33185455
REFERENCE   2  (bases 1 to 8249)
  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: "J Proteome 
            Res. 2020 Nov 13. doi: 10.1021/acs.jproteome.0c00616."
COMMENT     Sequence Label: pEF6-Lck-TurboID
FEATURES             Location/Qualifiers
     source          1..8249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1196..1215
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1196..1214
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2821..3777
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             3784..3810
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             3817..3843
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3866..3883)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3872..4096
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4142..4570
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4229..4248)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4439..4460
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4579..4599)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4584..4913
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4764..4899
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4826..4845
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4961..5008
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5027..5425
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5583..5704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5620..5639)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5674..5693
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5753..5769)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5753..5769)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5766..5788)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5777..5793
                     /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(5801..5807,5808..5825,5826..5831))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5846..5867
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5984..6001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6144..6732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6224..6243)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6903..7694,7695..7763))
                     /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"
     promoter        complement(7764..7868)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7943..7962)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaattagctt ggtactaata
     1201 cgactcacta tagggagacc caagctggct aggtaagctt ggtaccgagc tcggatccac
     1261 tagtccagtg tggtggaatt cgccatgggc tgtggctgca gctcacaccc ggaagatgac
     1321 tggatggaaa acatcgatgt gtgtgagaac tgccattatc ccatagtccc actggatggc
     1381 aagggcacgc tgctcatccg aaatggctct gaggtgcggg acccactggt tacctacgaa
     1441 ggctccaatc cgccggcttc cccactgcaa gacaacctgg ttatcgctct gcacagctat
     1501 gagccctctc acgacggaga tctgggcttt gagaaggggg aacagctccg catcctggag
     1561 cagagcggcg agtggtggaa ggcgcagtcc ctgaccacgg gccaggaagg cttcatcccc
     1621 ttcaattttg tggccaaagc gaacagcctg gagcccgaac cctggttctt caagaacctg
     1681 agccgcaagg acgcggagcg gcagctcctg gcgcccggga acactcacgg ctccttcctc
     1741 atccgggaga gcgagagcac cgcgggatcg ttttcactgt cggtccggga cttcgaccag
     1801 aaccagggag aggtggtgaa acattacaag atccgtaatc tggacaacgg tggcttctac
     1861 atctcccctc gaatcacttt tcccggcctg catgaactgg tccgccatta caccaatgct
     1921 tcagatgggc tgtgcacacg gttgagccgc ccctgccaga cccagaagcc ccagaagccg
     1981 tggtgggagg acgagtggga ggttcccagg gagacgctga agctggtgga gcggctgggg
     2041 gctggacagt tcggggaggt gtggatgggg tactacaacg ggcacacgaa ggtggcggtg
     2101 aagagcctga agcagggcag catgtccccg gacgccttcc tggccgaggc caacctcatg
     2161 aagcagctgc aacaccagcg gctggttcgg ctctacgctg tggtcaccca ggagcccatc
     2221 tacatcatca ctgaatacat ggagaatggg agtctagtgg attttctcaa gaccccttca
     2281 ggcatcaagt tgaccatcaa caaactcctg gacatggcag cccaaattgc agaaggcatg
     2341 gcattcattg aagagcggaa ttatattcat cgtgaccttc gggctgccaa cattctggtg
     2401 tctgacaccc tgagctgcaa gattgcagac tttggcctag cacgcctcat tgaggacaac
     2461 gagtacacag ccagggaggg ggccaagttt cccattaagt ggacagcgcc agaagccatt
     2521 aactacggga cattcaccat caagtcagat gtgtggtctt ttgggatcct gctgacggaa
     2581 attgtcaccc acggccgcat cccttaccca gggatgacca acccggaggt gattcagaac
     2641 ctggagcgag gctaccgcat ggtgcgccct gacaactgtc cagaggagct gtaccaactc
     2701 atgaggctgt gctggaagga gcgcccagag gaccggccca cctttgacta cctgcgcagt
     2761 gtgctggagg acttcttcac ggccacagag ggccagtacc agcctcagcc tggagctagc
     2821 aaagacaata ctgtgcctct gaagctgatc gctctcctgg ctaatggcga gttccatagt
     2881 ggcgaacagc tgggagaaac cctgggcatg tccagggccg ctatcaacaa gcacattcag
     2941 actctgcgcg actggggcgt ggacgtgttc accgtgcccg gaaagggcta ctctctgccc
     3001 gagcctatcc cgctgctgaa cgctaaacag attctgggac agctggacgg cgggagcgtg
     3061 gcagtcctgc ctgtggtcga ctccaccaat cagtacctgc tggatcgaat cggcgagctg
     3121 aagagtgggg atgcttgcat tgcagaatat cagcaggcag ggagaggaag cagagggagg
     3181 aaatggttct ctccttttgg agctaacctg tacctgagta tgttttggcg cctgaagcgg
     3241 ggaccagcag caatcggcct gggcccggtc atcggaattg tcatggcaga agcgctgcga
     3301 aagctgggag cagacaaggt gcgagtcaaa tggcccaatg acctgtatct gcaggataga
     3361 aagctggcag gcatcctggt ggagctggcc ggaataacag gcgatgctgc acagatcgtc
     3421 attggcgccg ggattaacgt ggctatgagg cgcgtggagg aaagcgtggt caatcagggc
     3481 tggatcacac tgcaggaagc agggattaac ctggacagga atactctggc cgctacgctg
     3541 atccgagagc tgcgggcagc cctggaactg ttcgagcagg aaggcctggc tccatatctg
     3601 ccacggtggg agaagctgga taacttcatc aatagacccg tgaagctgat cattggggac
     3661 aaagagattt tcgggattag ccgggggatt gataaacagg gagccctgct gctggaacag
     3721 gacggagtta tcaaaccctg gatgggcgga gaaatcagtc tgcggtctgc cgaaaagctg
     3781 cagctgcctc ccctggagcg cctgaccctg gacggatacc catacgatgt tccagattac
     3841 gcttaagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     3901 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     3961 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     4021 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     4081 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     4141 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4201 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4261 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4321 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4381 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4441 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4501 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4561 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     4621 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     4681 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4741 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4801 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     4861 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     4921 cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc
     4981 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagccttt
     5041 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat
     5101 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg
     5161 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc
     5221 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg
     5281 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa
     5341 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc
     5401 ctctggttat gtgtgggagg gctaagcact tcgtggccga ggagcaggac tgacacgtgc
     5461 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5521 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5581 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5641 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5701 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5761 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5821 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5881 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5941 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6001 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6061 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc caggaaccgt
     6121 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6181 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6241 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6301 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6361 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6421 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6481 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6541 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6601 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6661 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6721 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6781 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6841 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6901 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6961 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     7021 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7081 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7141 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7201 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7261 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7321 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7381 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7441 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7501 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7561 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7621 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7681 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7741 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7801 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7861 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     7921 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     7981 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     8041 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     8101 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact aggcttttgc
     8161 aaaaagcttt gcaaagatgg ataaagtttt aaacagagag gaatctttgc agctaatgga
     8221 ccttctaggt cttgaaagga gtgggaatt
//
