LOCUS       sequence-173579-        4953 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Akt pseudosubstrate 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 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: pHaloTag-PS
FEATURES             Location/Qualifiers
     source          1..4953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      13..601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     502..521
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        776..1079
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1080..1283
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1234..1254
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1315..1324
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1321..2211
                     /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    2456..2577
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2493..2512)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2547..2566
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2584..3039)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2721..2742)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2933..2952
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3066..3170
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3172..3529
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3172..3363
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3195..3215)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3380..3515
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3564..4358
                     /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             3564..4358
                     /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(3618..3637)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4228..4247
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4546..4565)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4590..4637
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
       61 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      121 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
      181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
      601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
      661 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt
      721 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      781 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      841 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      901 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      961 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1021 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     1081 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     1141 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1201 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     1261 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag caaagccacc
     1321 atggcagaaa tcggtactgg ctttccattc gacccccatt atgtggaagt cctgggcgag
     1381 cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt
     1441 aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc gacccatcgc
     1501 tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct gggttatttc
     1561 ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct ggaagaggtc
     1621 gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa gcgcaatcca
     1681 gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac ctgggacgaa
     1741 tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt cggccgcaag
     1801 ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg
     1861 ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag
     1921 ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa catcgtcgcg
     1981 ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct gctgttctgg
     2041 ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa aagcctgcct
     2101 aactgcaagg ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga caacccggac
     2161 ctgatcggca gcgagatcgc gcgctggctg tcgacgctcg agatttccgg ctccggactc
     2221 agatctcgag ctgttgaact agatcctgag tttgaacctc gtgccagaga gcgtgcttac
     2281 gccttcggac actaagaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggatc
     2341 tagataactg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     2401 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     2461 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     2521 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg
     2581 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2641 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2701 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2761 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2821 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2881 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2941 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3001 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     3061 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3121 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg
     3181 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3241 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3301 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3361 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3421 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     3481 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag
     3541 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3601 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3661 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3721 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     3781 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3841 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3901 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3961 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4021 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4081 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4141 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4201 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4261 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4321 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4381 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4441 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4501 ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga
     4561 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     4621 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     4681 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     4741 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     4801 agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4861 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4921 gttccactga gcgtcagacc ccgtagaaaa gat
//
