LOCUS       Exported                6728 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses human full-length Akt1(K14A) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    pFBD-Akt1(K14A)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6728)
  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 6728)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6728
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      10..598
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     499..518
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     752..769
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  903..1127
                     /mobile_element_type="transposon:Tn7"
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(1194..1727)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(1916..1944)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    2452..2500
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2525..2544
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(2636..2745)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        2769..2860
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     2785..2802
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             2909..2938
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2945..2968
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2978..2998
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     CDS             3002..4441
                     /codon_start=1
                     /gene="AKT1"
                     /product="human serine/threonine protein kinase activated 
                     by growth factors"
                     /label=Akt1
                     /translation="MSDVAIVKEGWLHARGEYIKTWRPRYFLLKNDGTFIGYKERPQDV
                     DQREAPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQ
                     TVADGLKKQEEEEMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGK
                     VILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDR
                     LCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLM
                     LDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMY
                     EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDA
                     KEIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSM
                     ECVDSERRPHFPQFSYSASSTA"
     polyA_signal    4610..4744
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4647..4666)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4701..4720
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  4773..4938
                     /mobile_element_type="transposon:Tn7"
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      5122..5576
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5209..5228)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5418..5439
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5602..5706
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5707..6567
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5925..5944)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
       61 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
      121 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
      181 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
      241 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
      301 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
      361 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
      421 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
      481 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      541 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      601 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      661 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      721 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      781 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagacca
      841 gccgcgtaac ctggcaaaat cggttacggt tgagtaataa atggatgccc tgcgtaagcg
      901 ggtgtgggcg gacaataaag tcttaaactg aacaaaatag atctaaacta tgacaataaa
      961 gtcttaaact agacagaata gttgtaaact gaaatcagtc cagttatgct gtgaaaaagc
     1021 atactggact tttgttatgg ctaaagcaaa ctcttcattt tctgaagtgc aaattgcccg
     1081 tcgtattaaa gaggggcgtg gccaagggca tggtaaagac tatattcgcg gcgttgtgac
     1141 aatttaccga acaactccgc ggccgggaag ccgatctcgg cttgaacgaa ttgttaggtg
     1201 gcggtacttg ggtcgatatc aaagtgcatc acttcttccc gtatgcccaa ctttgtatag
     1261 agagccactg cgggatcgtc accgtaatct gcttgcacgt agatcacata agcaccaagc
     1321 gcgttggcct catgcttgag gagattgatg agcgcggtgg caatgccctg cctccggtgc
     1381 tcgccggaga ctgcgagatc atagatatag atctcactac gcggctgctc aaacttgggc
     1441 agaacgtaag ccgcgagagc gccaacaacc gcttcttggt cgaaggcagc aagcgcgatg
     1501 aatgtcttac tacggagcaa gttcccgagg taatcggagt ccggctgatg ttgggagtag
     1561 gtggctacgt ctccgaactc acgaccgaaa agatcaagag cagcccgcat ggatttgact
     1621 tggtcagggc cgagcctaca tgtgcgaatg atgcccatac ttgagccacc taactttgtt
     1681 ttagggcgac tgccctgctg cgtaacatcg ttgctgctgc gtaacatcgt tgctgctcca
     1741 taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc gaggcataga
     1801 ctgtacaaaa aaacagtcat aacaagccat gaaaaccgcc actgcgccgt taccaccgct
     1861 gcgttcggtc aaggttctgg accagttgcg tgagcgcata cgctacttgc attacagttt
     1921 acgaaccgaa caggcttatg tcaactgggt tcgtgccttc atccgtttcc acggtgtgcg
     1981 tcacccggca accttgggca gcagcgaagt cgaggcattt ctgtcctggc tggcgaacga
     2041 gcgcaaggtt tcggtctcca cgcatcgtca ggcattggcg gccttgctgt tcttctacgg
     2101 caaggtgctg tgcacggatc tgcccttgct tcaggagatc ggtagacctc ggccgtcgcg
     2161 gcgcttgccg gtggtgctga ccccggatga agtggttcgc atcctcggtt ttctggaagg
     2221 cgagcatcgt ttgttcgccc aggactctag ctatagttct agtggttggc tacgtacccg
     2281 tagtggctat ggcagggctt gccgccccga cgttggctgc gagccctggg ccttcacccg
     2341 aacttggggg ttggggtggg gaaaaggaag aaacgcgggc gtattggtcc caatggggtc
     2401 tcggtggggt atcgacagag tgccagccct gggaccgaac cccgcgttta tgaacaaacg
     2461 acccaacacc cgtgcgtttt attctgtctt tttattgccg tcatagcgcg ggttccttcc
     2521 ggtattgtct ccttccgtgt ttcagttagc ctcccccatc tcccggtacc gcatgctatg
     2581 catcagctgc tagcaccatg gctcgagatc ccgggtgatc aagtcttcgt cgagtgattg
     2641 taaataaaat gtaatttaca gtatagtatt ttaattaata tacaaatgat ttgataataa
     2701 ttcttattta actataatat attgtgttgg gttgaattaa aggtccgtat actccggaat
     2761 attaatagat catggagata attaaaatga taaccatctc gcaaataaat aagtatttta
     2821 ctgttttcgt aacagttttg taataaaaaa acctataaat attccggatt attcataccg
     2881 tcccaccatc gggcgcggat ccatgtcgca ccatcaccat caccatcacc atcaccatgc
     2941 aagctggagc cacccgcagt tcgaaaaggg ctccggtgaa aacctgtatt tccaggcgcg
     3001 catgagcgac gtggctattg tgaaggaggg ttggctgcac gcacgagggg agtacatcaa
     3061 gacctggcgg ccacgctact tcctcctcaa gaatgatggc accttcattg gctacaagga
     3121 gaggccgcag gatgtggacc aacgtgaggc tcccctcaac aacttctctg tggcgcagtg
     3181 ccagctgatg aagacggagc ggccccggcc caacaccttc atcatccgct gcctgcagtg
     3241 gaccactgtc atcgaacgca ccttccatgt ggagactcct gaggagcggg aggagtggac
     3301 aaccgccatc cagactgtgg ctgacggcct caagaagcag gaggaggagg agatggactt
     3361 ccggtcgggc tcacccagtg acaactcagg ggctgaagag atggaggtgt ccctggccaa
     3421 gcccaagcac cgcgtgacca tgaacgagtt tgagtacctg aagctgctgg gcaagggcac
     3481 tttcggcaag gtgatcctgg tgaaggagaa ggccacaggc cgctactacg ccatgaagat
     3541 cctcaagaag gaagtcatcg tggccaagga cgaggtggcc cacacactca ccgagaaccg
     3601 cgtcctgcag aactccaggc accccttcct cacagccctg aagtactctt tccagaccca
     3661 cgaccgcctc tgctttgtca tggagtacgc caacgggggc gagctgttct tccacctgtc
     3721 ccgggagcgt gtgttctccg aggaccgggc ccgcttctat ggcgctgaga ttgtgtcagc
     3781 cctggactac ctgcactcgg agaagaacgt ggtgtaccgg gacctcaagc tggagaacct
     3841 catgctggac aaggacgggc acattaagat cacagacttc gggctgtgca aggaggggat
     3901 caaggacggt gccaccatga agaccttttg cggcacacct gagtacctgg cccccgaggt
     3961 gctggaggac aatgactacg gccgtgcagt ggactggtgg gggctgggcg tggtcatgta
     4021 cgagatgatg tgcggtcgcc tgcccttcta caaccaggac catgagaagc tttttgagct
     4081 catcctcatg gaggagatcc gcttcccgcg cacgcttggt cccgaggcca agtccttgct
     4141 ttcagggctg ctcaagaagg accccaagca gaggcttggc gggggctccg aggacgccaa
     4201 ggagatcatg cagcatcgct tctttgccgg tatcgtgtgg cagcacgtgt acgagaagaa
     4261 gctcagccca cccttcaagc cccaggtcac gtcggagact gacaccaggt attttgatga
     4321 ggagttcacg gcccagatga tcaccatcac accacctgac caagatgaca gcatggagtg
     4381 tgtggacagc gagcgcaggc cccacttccc ccagttctcc tactcggcca gcagcacggc
     4441 ctgagcggcc gctttcgaat ctagagcctg cagtctcgac aagcttgtcg agaagtacta
     4501 gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     4561 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     4621 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4681 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4741 gatctgatca ctgcttgagc ctaggagatc cgaaccagat aagtgaaatc tagttccaaa
     4801 ctattttgtc atttttaatt ttcgtattag cttacgacgc tacacccagt tcccatctat
     4861 tttgtcactc ttccctaaat aatccttaaa aactccattt ccacccctcc cagttcccaa
     4921 ctattttgtc cgcccacagc ggggcatttt tcttcctgtt atgtttttaa tcaaacatcc
     4981 tgccaactcc atgtgacaaa ccgtcatctt cggctacttt ttctctgtca cagaatgaaa
     5041 atttttctgt catctcttcg ttattaatgt ttgtaattga ctgaatatca acgcttattt
     5101 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     5161 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     5221 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5281 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     5341 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5401 agtccacgtt cttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5461 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5521 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     5581 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5641 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5701 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5761 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5821 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5881 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5941 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     6001 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6061 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6121 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6181 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6241 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6301 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6361 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6421 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6481 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6541 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6601 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6661 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6721 aagatcaa
//
