LOCUS       sequence-174208-        5193 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses eGFP-taggd PH domain of Btk.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5193)
  AUTHORS   Ebner M, Lucic I, Leonard TA, Yudushkin I
  TITLE     PI(3,4,5)P3 Engagement Restricts Akt Activity to Cellular Membranes.
  JOURNAL   Mol Cell. 2017 Feb 2;65(3):416-431.e6. doi: 
            10.1016/j.molcel.2016.12.028.
  PUBMED    28157504
REFERENCE   2  (bases 1 to 5193)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2016.12"; journalName: "Mol Cell"; date: 
            "2017-02-2- 2"; volume: "65"; issue: "3"; pages: "416-431.e6"
COMMENT     Sequence Label: pEGFP-PH(Btk)
FEATURES             Location/Qualifiers
     source          1..5193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      16..604
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     505..524
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        779..1082
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1083..1286
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1237..1257
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1325..1334
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1331..1333,1334..1336,1337..2047)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1376..1397)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1637..1656)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1984..2005
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2699..2820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2736..2755)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2790..2809
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2827..3282)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2964..2985)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3176..3195
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3309..3413
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3415..3772
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3415..3606
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3438..3458)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3623..3758
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3807..4601
                     /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             3807..4601
                     /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(3861..3880)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4471..4490
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4789..4808)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4833..4880
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
       61 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
      121 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
      181 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
      241 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
      301 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
      361 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
      421 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
      481 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
      541 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
      601 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
      661 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta
      721 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      781 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      841 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      901 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      961 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     1021 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     1081 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     1141 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     1201 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     1261 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc
     1321 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1381 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1441 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1501 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1561 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1621 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1681 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1741 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1801 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1861 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1921 cgacaaccac tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga
     1981 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2041 gtacaagtcc ggactcagat ctcgagctat ggccgcagtg attctggaga gcatctttct
     2101 gaagcgatcc caacagaaaa agaaaacatc acctctaaac ttcaagaagc gcctgtttct
     2161 cttgaccgtg cacaaactct cctactatga gtatgacttt gaacgtggga gaagaggcag
     2221 taagaagggt tcaatagatg ttgagaagat cacttgtgtt gaaacagtgg ttcctgaaaa
     2281 aaatcctcct ccagaaagac agattccgag aagaggtgaa gagtccagtg aaatggagca
     2341 aatttcaatc attgaaaggt tcccttatcc cttccaggtt gtatatgatg aagggcctct
     2401 ctacgtcttc tccccaactg aagaactaag gaagcggtgg attcaccagc tcaaaaacgt
     2461 aatccggtac aacagtgatc tggttcagaa atatcaccct tgcttctgga tcgatgggca
     2521 gtatctctgc tgctctcaga cagccaaaaa tgctatgggc tgccaaattt tggagaactg
     2581 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     2641 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     2701 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2761 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2821 acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2881 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2941 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     3001 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     3061 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     3121 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     3181 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     3241 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     3301 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3361 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     3421 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3481 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3541 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3601 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3661 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     3721 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     3781 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3841 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3901 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3961 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     4021 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4081 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4141 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4201 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4261 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4321 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4381 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4441 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4501 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4561 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     4621 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4681 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4741 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     4801 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     4861 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     4921 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     4981 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     5041 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5101 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5161 ttcgttccac tgagcgtcag accccgtaga aaa
//
