LOCUS       sequence-173581-        4953 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Akt pseudosubstrate (P-3 RA) mutant tagged with a HaloTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4953)
  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 4953)
  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: 
            "10.1016/j.molcel.2016.12"; journalName: "Mol Cell"; date: 
            "2017-02-2- 2"; volume: "65"; issue: "3"; pages: "416-431.e6"
COMMENT     Sequence Label: pHaloTag-PS(RA)
FEATURES             Location/Qualifiers
     source          1..4953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      21..609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     510..529
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        784..1087
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1088..1291
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1242..1262
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1323..1332
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1329..2219
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     polyA_signal    2464..2585
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2501..2520)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2555..2574
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2592..3047)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2729..2750)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2941..2960
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3074..3178
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3180..3537
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3180..3371
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3203..3223)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3388..3523
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3572..4366
                     /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             3572..4366
                     /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(3626..3645)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4236..4255
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4554..4573)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4598..4645
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
       61 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
      121 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
      181 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
      241 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
      301 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
      361 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
      421 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
      481 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
      541 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
      601 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
      661 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
      721 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      781 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      841 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      901 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      961 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     1021 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     1081 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     1141 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     1201 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     1261 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagca
     1321 aagccaccat ggcagaaatc ggtactggct ttccattcga cccccattat gtggaagtcc
     1381 tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc
     1441 tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga
     1501 cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg
     1561 gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg
     1621 aagaggtcgt cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc
     1681 gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct
     1741 gggacgaatg gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg
     1801 gccgcaagct gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg
     1861 tccgcccgct gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg
     1921 accgcgagcc actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca
     1981 tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc
     2041 tgttctgggg caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa
     2101 gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca
     2161 acccggacct gatcggcagc gagatcgcgc gctggctgtc gacgctcgag atttccggct
     2221 ccggactcag atctcgagct gttgaactag atcctgagtt tgaacctcgt gccgcagagc
     2281 gtgcttacgc cttcggacac taagaattct gcagtcgacg gtaccgcggg cccgggatcc
     2341 accggatcta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     2401 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     2461 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2521 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2581 tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2641 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2701 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2761 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2821 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2881 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2941 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     3001 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     3061 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3121 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     3181 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3241 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3301 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3361 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3421 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     3481 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     3541 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     3601 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3661 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3721 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     3781 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3841 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3901 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3961 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4021 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4081 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4141 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4201 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4261 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4321 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4381 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4441 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4501 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     4561 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     4621 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     4681 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     4741 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     4801 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     4861 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4921 gagttttcgt tccactgagc gtcagacccc gta
//
