LOCUS       sequence-486042-        6807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Lyn-FRB_star and 
            mVenus-FKBP-5ptase(phosphatase-dead mutant) of INPP5E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6807)
  AUTHORS   Obashi K, Strub MP, Taraska JW
  TITLE     A PIP2-stabilized syntaxin-1a structure mapped with transition metal
            ion FRET and unnatural fluorescent amino acids at the plasma 
            membrane.
  JOURNAL   Structure. 2025 Nov 24:S0969-2126(25)00388-0. doi: 
            10.1016/j.str.2025.10.001.
  PUBMED    41290003
REFERENCE   2  (bases 1 to 6807)
  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: "Structure. 
            2025 Nov 24:S0969-2126(25)00388-0. doi: 10.1016/j.str.2025.10.001."
COMMENT     Sequence Label: Lyn-FRB_star-P2A-mVenus-FKBP-5ptase(D343A)
FEATURES             Location/Qualifiers
     source          1..6807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      637..646
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             712..990
                     /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             1000..1056
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(1057..1059,1060..1770)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1099..1120)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1360..1379)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1707..1728
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1831..2151
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     polyA_signal    3595..3716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3632..3651)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3686..3705
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3723..4178)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3860..3881)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4072..4091
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4205..4309
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4311..4668
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4311..4502
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4334..4354)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4519..4654
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4703..5497
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4703..5497
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4757..4776)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5367..5386
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5685..5704)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5729..5776
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6105..6693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6594..6613
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attccggcca ccatgggatg tataaaatca
      661 aaagggaaag acagcgcgga tccaccggtc gccaccaagg gagcaagtgg aatcctctgg
      721 catgagatgt ggcatgaagg cctggaagag gcatctcgtt tgtactttgg ggaaaggaac
      781 gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta tgatggaacg gggcccccag
      841 actctgaagg aaacatcctt taatcaggcc tatggtcgag atttaatgga ggcccaagag
      901 tggtgcagga agtacatgaa atcagggaat gtcaaggacc tcctccaagc ctgggacctc
      961 tattatcatg tgttccgacg aatctcaaag ggaagcggag ctactaactt cagcctgctg
     1021 aagcaggctg gcgacgtgga ggagaaccct ggacctgtga gcaagggcga ggagctgttc
     1081 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1141 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gctgatctgc
     1201 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctggg ctacggcctg
     1261 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1321 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1381 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1441 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1501 aacgtctata tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc
     1561 cacaacatcg aggacggcgg cgtgcagctc gccgaccact accagcagaa cacccccatc
     1621 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc caagctgagc
     1681 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1741 atcactctcg gcatggacga gctgtacaag tccggactca gatctcgaag cgcggccgcg
     1801 ggagcaggag gagcagctcg agcggcgctg ggagtgcagg tggaaaccat ctccccagga
     1861 gacgggcgca ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt
     1921 gaagatggaa agaaatttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta
     1981 ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag
     2041 agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc
     2101 atcccaccac atgccactct cgtcttcgat gtggagcttc taaaactgga agctcgaggc
     2161 gcagcagcgg gagcaggagg agcaggtcga tcggatcttg cagactacaa gctccgcgcg
     2221 cagccgctcc tggtgcgggc ccacagcagc ctgggccccg gccggccgcg gagccccctg
     2281 gcctgcgacg actgttccct tcgctcggcc aaatcctcct tcagcctcct ggcgcccatc
     2341 cgcagcaagg acgtccgcag caggagttac ctggagggca gcctcctggc cagcggggcc
     2401 ctgttggggg cggatgagct ggcccgctac ttcccagacc ggaacgtggc actcttcgtg
     2461 gccacctgga acatgcaggg ccagaaggag ctcccgccca gcctggacga gttcctgctc
     2521 ccagccgagg ccgactatgc ccaggacctg tatgtcatcg gggtccagga gggctgttct
     2581 gacaggcggg agtgggagac tcgtctgcag gagacgctgg gcccgcacta tgtgctgctg
     2641 tcctcggcgg cccacggcgt gctctacatg tcgctcttca tccgcaggga cctcatctgg
     2701 ttctgctcag aggtggagtg ctccacggtg accacacgca tcgtgtctca gatcaagacc
     2761 aagggggcct tgggcatcag cttcaccttt tttggcactt ccttcctctt catcacgtcc
     2821 cacttcacct caggtgacgg gaaggtggcg gagcggctgc tggactacac caggactgta
     2881 caagccctgg tcctgcccag aaatgtgccc gacaccaacc cctatcgctc cagcgcagcg
     2941 gacgtcacca cccgcttcga tgaggtgttc tggtttggag acttcaactt ccgcctgagt
     3001 ggcgggcgca cagtcgtgga cgccctcctg tgccagggcc tggtggtgga cgtgccggcg
     3061 ctgctgcagc acgaccagct catccgggag atgcggaaag ggtccatctt caagggcttc
     3121 caggagccgg acatccactt cctcccatca tacaagtttg acatcgggaa ggacacgtac
     3181 gacagcacct ccaagcagag gacgccctca tacacggccc gcgtcttgta cagaagccgc
     3241 cacaagggtg acatctgtcc tgtgagctac tcttcctgcc ccgggatcaa gacgtccgac
     3301 caccgccctg tgtatggcct cttccgggtg aaagtgaggc cggggcgaga caacattccg
     3361 ttggcagctg gcaaatttga tagagaactg tacttactag gaattaaaag acggatttcg
     3421 aaggagattc agaggcagca agcactacag agtcagaact ccagcaccat cgcctccgtt
     3481 tcttaactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3541 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3601 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3661 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     3721 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3781 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3841 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3901 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3961 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     4021 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4081 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4141 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     4201 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4261 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     4321 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4381 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4441 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4501 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4561 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4621 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4681 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4741 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4801 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4861 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4921 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4981 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     5041 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5101 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5161 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5221 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5281 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5341 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5401 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5461 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5521 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5581 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5641 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5701 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5761 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     5821 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     5881 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     5941 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6001 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6061 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6121 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6181 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6241 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtatca
     6301 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6361 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6421 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6481 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6541 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6601 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6661 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6721 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6781 gtggataacc gtattaccgc catgcat
//
