LOCUS       sequence-174210-        6273 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses mCherry-tagged chimeric fusion of Btk PH domain with the 
            kinase domain of human Akt1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6273)
  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 6273)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 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: pmCherry-PH(Btk)KD(Akt1)
FEATURES             Location/Qualifiers
     source          1..6273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     500..519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        774..1077
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1078..1281
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1232..1252
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1320..1329
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1326..2033
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1471..1489)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1739..1758
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3774..3895
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3811..3830)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3865..3884
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3902..4357)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4039..4060)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4251..4270
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4384..4488
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4490..4847
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4490..4681
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4513..4533)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4698..4833
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4882..5676
                     /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             4882..5676
                     /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(4936..4955)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5546..5565
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5864..5883)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5908..5955
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
       61 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
      121 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
      181 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
      241 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
      301 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
      361 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      421 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      481 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      541 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      601 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      661 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat
      721 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      781 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      841 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      901 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      961 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     1021 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     1081 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     1141 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     1201 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     1261 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg
     1321 ccaccatggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     1381 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     1441 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     1501 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     1561 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     1621 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     1681 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     1741 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     1801 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     1861 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     1921 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     1981 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtccggac
     2041 tcagatctcg agctatggcc gcagtgattc tggagagcat ctttctgaag cgatcccaac
     2101 agaaaaagaa aacatcacct ctaaacttca agaagcgcct gtttctcttg accgtgcaca
     2161 aactctccta ctatgagtat gactttgaac gtgggagaag aggcagtaag aagggttcaa
     2221 tagatgttga gaagatcact tgtgttgaaa cagtggttcc tgaaaaaaat cctcctccag
     2281 aaagacagat tccgagaaga ggtgaagagt ccagtgaaat ggagcaaatt tcaatcattg
     2341 aaaggttccc ttatcccttc caggttgtat atgatgaagg gcctctctac gtcttctccc
     2401 caactgaaga actaaggaag cggtggattc accagctcaa aaacgtaatc cggtacaaca
     2461 gtgatctggt tcagaaatat cacccttgct tctggatcga tgggcagtat ctctgctgct
     2521 ctcagacagc caaaaatgct atgggctgcc aaattttgga gaacatggac ttccggtcgg
     2581 gctcacccag tgacaactca ggggctgaag agatggaggt gtccctggcc aagcccaagc
     2641 accgcgtgac catgaacgag tttgagtacc tgaagctgct gggcaagggc actttcggca
     2701 aggtgatcct ggtgaaggag aaggccacag gccgctacta cgccatgaag atcctcaaga
     2761 aggaagtcat cgtggccaag gacgaggtgg cccacacact caccgagaac cgcgtcctgc
     2821 agaactccag gcaccccttc ctcacagccc tgaagtactc tttccagacc cacgaccgcc
     2881 tctgctttgt catggagtac gccaacgggg gcgagctgtt cttccacctg tcccgggagc
     2941 gtgtgttctc cgaggaccgg gcccgcttct atggcgctga gattgtgtca gccctggact
     3001 acctgcactc ggagaagaac gtggtgtacc gggacctcaa gctggagaac ctcatgctgg
     3061 acaaggacgg gcacattaag atcacagact tcgggctgtg caaggagggg atcaaggacg
     3121 gtgccaccat gaagaccttt tgcggcacac ctgagtacct ggcccccgag gtgctggagg
     3181 acaatgacta cggccgtgca gtggactggt gggggctggg cgtggtcatg tacgagatga
     3241 tgtgcggtcg cctgcccttc tacaaccagg accatgagaa gctttttgag ctcatcctca
     3301 tggaggagat ccgcttcccg cgcacgcttg gtcccgaggc caagtccttg ctttcagggc
     3361 tgctcaagaa ggaccccaag cagaggcttg gcgggggctc cgaggacgcc aaggagatca
     3421 tgcagcatcg cttctttgcc ggtatcgtgt ggcagcacgt gtacgagaag aagctcagcc
     3481 cacccttcaa gccccaggtc acgtcggaga ctgacaccag gtattttgat gaggagttca
     3541 cggcccagat gatcaccatc acaccacctg accaagatga cagcatggag tgtgtggaca
     3601 gcgagcgcag gccccacttc ccccagttct cctactcggc cagcggcacg gcctgatcta
     3661 gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     3721 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     3781 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3841 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg
     3901 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     3961 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     4021 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     4081 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     4141 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     4201 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     4261 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     4321 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     4381 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4441 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     4501 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     4561 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     4621 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     4681 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4741 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     4801 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     4861 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4921 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4981 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5041 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     5101 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5161 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5221 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5281 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5341 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5401 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5461 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5521 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5581 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5641 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5701 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5761 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5821 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     5881 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     5941 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     6001 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     6061 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     6121 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6181 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6241 tccactgagc gtcagacccc gtagaaaaga tca
//
