LOCUS       sequence-350569-        8255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Baculo expression of decahistidine tag fused to 4xFRB, 1xSNAP tag 
            and human Lck G2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8255)
  AUTHORS   Hui E, Vale RD
  TITLE     In vitro membrane reconstitution of the T-cell receptor proximal 
            signaling network.
  JOURNAL   Nat Struct Mol Biol. 2014 Feb;21(2):133-42. doi: 10.1038/nsmb.2762. 
            Epub 2014 Jan 26.
  PUBMED    24463463
REFERENCE   2  (bases 1 to 8255)
  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: "10.1038/nsmb.2762";
            journalName: "Nat Struct Mol Biol"; date: "2014-02"; volume: "21"; 
            issue: "2"; pages: "133-42"
COMMENT     Sequence Label: pFastBacHT (His10-4xFRB-SNAP-Lck G2A)
FEATURES             Location/Qualifiers
     source          1..8255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2360..2377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2511..2735
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2802..3335)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3505..3510,3511..3527,3528..3533))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        3885..3976
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     3901..3918
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             4043..4072
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4163..4441
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             4484..4762
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             4805..5083
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             5126..5404
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             5447..5992
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     polyA_signal    7745..7879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7782..7801)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7836..7855
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7908..8073
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgtg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct
     2461 ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga
     2521 caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag
     2581 acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt
     2641 tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga
     2701 ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac
     2761 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     2821 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     2881 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     2941 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3001 gcgagatcat agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc
     3061 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3121 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3181 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3241 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3301 ccctgctgcg taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc
     3361 gcttgctgct tggatgcccg aggcatagac tgtacaaaaa aacagtcata acaagccatg
     3421 aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt
     3481 gagcgcatac gctacttgca ttacagttta cgaaccgaac aggcttatgt caactgggtt
     3541 cgtgccttca tccgtttcca cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc
     3601 gaggcatttc tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag
     3661 gcattggcgg ccttgctgtt cttctacggc aaggtgctgt gcacggatct gccctggctt
     3721 caggagatcg gaagacctcg gccgtcgcgg cgcttgccgg tggtgctgac cccggatgaa
     3781 gtggttcgca tcctcggttt tctggaaggc gagcatcgtt tgttcgccca ggactctagc
     3841 tatagttcta gtggttggct acgtatactc cggaatatta atagatcatg gagataatta
     3901 aaatgataac catctcgcaa ataaataagt attttactgt tttcgtaaca gttttgtaat
     3961 aaaaaaacct ataaatattc cggattattc ataccgtccc accatcgggc gcggatctcg
     4021 gtccgaaacc atgtcgtact accatcacca tcaccatcac catcaccatc acgattacga
     4081 tatcccaacg accgaaaacc ggtattttca gggcgccatg ggatccggaa ttcaaaggcc
     4141 tacgtcgacg agctcaacta gaatcctctg gcatgagatg tggcatgaag gcctggaaga
     4201 ggcatctcgt ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg tgctggagcc
     4261 cttgcatgct atgatggaac ggggccccca gactctgaag gaaacatcct ttaatcaggc
     4321 ctatggtcga gatttaatgg aggcccaaga gtggtgcagg aagtacatga aatcagggaa
     4381 tgtcaaggac ctcctccaag cctgggacct ctattatcat gtgttccgac gaatctcaaa
     4441 gggtagcggt agcggtagcg gtggaggtgg aagcagcact agaatcctct ggcatgagat
     4501 gtggcatgaa ggcctggaag aggcatctcg tttgtacttt ggggaaagga acgtgaaagg
     4561 catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc agactctgaa
     4621 ggaaacatcc tttaatcagg cctatggtcg agatttaatg gaggcccaag agtggtgcag
     4681 gaagtacatg aaatcaggga atgtcaagga cctcctccaa gcctgggacc tctattatca
     4741 tgtgttccga cgaatctcaa agggtagcgg tagcggtagc ggtggaggtg gaagcagcac
     4801 tagaatcctc tggcatgaga tgtggcatga aggcctggaa gaggcatctc gtttgtactt
     4861 tggggaaagg aacgtgaaag gcatgtttga ggtgctggag cccttgcatg ctatgatgga
     4921 acggggcccc cagactctga aggaaacatc ctttaatcag gcctatggtc gagatttaat
     4981 ggaggcccaa gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg acctcctcca
     5041 agcctgggac ctctattatc atgtgttccg acgaatctca aagggtagcg gtagcggtag
     5101 cggtggaggt ggaagcagca ctagaatcct ctggcatgag atgtggcatg aaggcctgga
     5161 agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg aggtgctgga
     5221 gcccttgcat gctatgatgg aacggggccc ccagactctg aaggaaacat cctttaatca
     5281 ggcctatggt cgagatttaa tggaggccca agagtggtgc aggaagtaca tgaaatcagg
     5341 gaatgtcaag gacctcctcc aagcctggga cctctattat catgtgttcc gacgaatctc
     5401 aaagggtagc ggtagcggta gcggtggagg tggaagcagc actagaatgg acaaagattg
     5461 cgaaatgaaa cgtaccaccc tggatagccc gctgggcaaa ctggaactga gcggctgcga
     5521 acagggcctg catgaaatta aactgctggg taaaggcacc agcgcggccg atgcggttga
     5581 agttccggcc ccggccgccg tgctgggtgg tccggaaccg ctgatgcagg cgaccgcgtg
     5641 gctgaacgcg tattttcatc agccggaagc gattgaagaa tttccggttc cggcgctgca
     5701 tcatccggtg tttcagcagg agagctttac ccgtcaggtg ctgtggaaac tgctgaaagt
     5761 ggttaaattt ggcgaagtga ttagctatca gcagctggcg gccctggcgg gtaatccggc
     5821 ggccaccgcc gccgttaaaa ccgcgctgag cggtaacccg gtgccgattc tgattccgtg
     5881 ccatcgtgtg gttagctcta gcggtgcggt tggcggttat gaaggtggtc tggcggtgaa
     5941 agagtggctg ctggcccatg aaggtcatcg tctgggtaaa ccgggtctgg gaggtagcgg
     6001 tagcggtagc ggtggaggtg gaagcagcac tagagcctgt ggctgcagct cacacccgga
     6061 agatgactgg atggaaaaca tcgatgtgtg tgagaactgc cattatccca tagtcccact
     6121 ggatggcaag ggcacgctgc tcatccgaaa tggctctgag gtgcgggacc cactggttac
     6181 ctacgaaggc tccaatccgc cggcttcccc actgcaagac aacctggtta tcgctctgca
     6241 cagctatgag ccctctcacg acggagatct gggctttgag aagggggaac agctccgcat
     6301 cctggagcag agcggcgagt ggtggaaggc gcagtccctg accacgggcc aggaaggctt
     6361 catccccttc aattttgtgg ccaaagcgaa cagcctggag cccgaaccct ggttcttcaa
     6421 gaacctgagc cgcaaggacg cggagcggca gctcctggcg cccgggaaca ctcacggctc
     6481 cttcctcatc cgggagagcg agagcaccgc gggatcgttt tcactgtcgg tccgggactt
     6541 cgaccagaac cagggagagg tggtgaaaca ttacaagatc cgtaatctgg acaacggtgg
     6601 cttctacatc tcccctcgaa tcacttttcc cggcctgcat gaactggtcc gccattacac
     6661 caatgcttca gatgggctgt gcacacggtt gagccgcccc tgccagaccc agaagcccca
     6721 gaagccgtgg tgggaggacg agtgggaggt tcccagggag acgctgaagc tggtggagcg
     6781 gctgggggct ggacagttcg gggaggtgtg gatggggtac tacaacgggc acacgaaggt
     6841 ggcggtgaag agcctgaagc agggcagcat gtccccggac gccttcctgg ccgaggccaa
     6901 cctcatgaag cagctgcaac accagcggct ggttcggctc tacgctgtgg tcacccagga
     6961 gcccatctac atcatcactg aatacatgga gaatgggagt ctagtggatt ttctcaagac
     7021 cccttcaggc atcaagttga ccatcaacaa actcctggac atggcagccc aaattgcaga
     7081 aggcatggca ttcattgaag agcggaatta tattcatcgt gaccttcggg ctgccaacat
     7141 tctggtgtct gacaccctga gctgcaagat tgcagacttt ggcctagcac gcctcattga
     7201 ggacaacgag tacacagcca gggagggggc caagtttccc attaagtgga cagcgccaga
     7261 agccattaac tacgggacat tcaccatcaa gtcagatgtg tggtcttttg gcatcctgct
     7321 gacggaaatt gtcacccacg gccgcatccc ttacccaggg atgaccaacc cggaggtgat
     7381 tcagaacctg gagcgaggct accgcatggt gcgccctgac aactgtccag aggagctgta
     7441 ccaactcatg aggctgtgct ggaaggagcg cccagaggac cggcccacct ttgactacct
     7501 gcgcagtgtg ctggaggact tcttcacggc cacagagggc cagtaccagc ctcagcctta
     7561 aactagtgcg gccgctttcg aatctagagc ctgcagtctc gaggcatgcg gtaccaagct
     7621 tgtcgagaag tactagagga tcataatcag ccataccaca tttgtagagg ttttacttgc
     7681 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     7741 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     7801 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     7861 atcttatcat gtctggatct gatcactgct tgagcctagg agatccgaac cagataagtg
     7921 aaatctagtt ccaaactatt ttgtcatttt taattttcgt attagcttac gacgctacac
     7981 ccagttccca tctattttgt cactcttccc taaataatcc ttaaaaactc catttccacc
     8041 cctcccagtt cccaactatt ttgtccgccc acagcggggc atttttcttc ctgttatgtt
     8101 tttaatcaaa catcctgcca actccatgtg acaaaccgtc atcttcggct actttttctc
     8161 tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt aatgtttgta attgactgaa
     8221 tatcaacgct tatttgcagc ctgaatggcg aatgg
//
