LOCUS       sequence-382462-        7413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre/LoxP-dependent expression of a constitutively active form of 
            Rheb (S16H). Expression of Cre excises a STOP codon before 
            Rheb(S16H), facilitating expression of a constitutively active form 
            of Rheb..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7413)
  AUTHORS   Lin TV, Hsieh L, Kimura T, Malone TJ, Bordey A
  TITLE     Normalizing translation through 4E-BP prevents mTOR-driven cortical 
            mislamination and ameliorates aberrant neuron integration.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Oct 4;113(40):11330-11335. Epub 2016 
            Sep 19.
  PUBMED    27647922
REFERENCE   2  (bases 1 to 7413)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2016-10-4"; volume: "113(40)"; pages: 
            "11330-11335. Epub 2016 Sep 19"
COMMENT     Sequence Label: pCALNL-Rheb(S16H)
FEATURES             Location/Qualifiers
     source          1..7413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          658..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1749..1782)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1832..2626
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1886..1905)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2496..2515
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2806..2940
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2954..2987)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3640..3649
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3646..3648,3649..3651,3652..4365)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3646..3648,3649..3651,3652..4362)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3691..3712)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3952..3971)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4299..4320
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4452..4471)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4517..4572
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4571..4590)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4933..4949)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4933..4949)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4946..4968)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4957..4973
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4981..4987,4988..5005,5006..5011))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5026..5047
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      5132..5266
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5286..5420
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5488..5505)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5659..6247)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5739..5758)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6418..7209,7210..7278))
                     /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     7041..7060
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7279..7383)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gggcgcgccr gggggggggg gggggggggg gggggggggg
      541 ggggsgrggs ggrgrggkgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      601 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      661 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      721 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      781 ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc
      841 cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg
      901 tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc
      961 gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
     1081 gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggrggg ctcgggggrg gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgat taattcgagc
     1741 gaacgcgtat aacttcgtat agcatacatt atacgaagtt atctcgagtc ggatttgatc
     1801 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     1861 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     1921 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     1981 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     2041 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     2101 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     2161 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     2221 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     2281 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     2341 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     2401 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     2461 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     2521 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     2581 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     2641 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     2701 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     2761 cagcgcgggg atctcatgct ggagttcttc gcccacccca tcgataactt gtttattgca
     2821 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     2881 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     2941 aatccgaacg cgtataactt cgtatagcat acattatacg aagttatctc gagtcgctcg
     3001 gtacgattta aattgaattc ctcgagatcc atgccgcagt ccaagtcccg gaagatcgcg
     3061 atcctgggct accggcatgt ggggaaatcc tcattgacga ttcaatttgt tgaaggccaa
     3121 tttgtggact cctacgatcc aaccatagaa aacactttta caaagttgat cacagtaaat
     3181 ggacaagaat atcatcttca acttgtagac acagccgggc aagatgaata ttctatcttt
     3241 cctcagacat actccataga tattaatggc tatattcttg tgtattctgt tacatcaatc
     3301 aaaagttttg aagtgattaa agttatccat ggcaaattgt tggatatggt ggggaaagta
     3361 caaataccta ttatgttggt tgggaataag aaagacctgc atatggaaag ggtgatcagt
     3421 tatgaagaag ggaaagcttt ggcagaatct tggaatgcag cttttttgga atcttctgct
     3481 aaagaaaatc agactgctgt ggatgttttt cgaaggataa ttttggaggc agaaaaaatg
     3541 gacggggcag cttcacaagg caagtcttca tgctcggtga tgtaactcga tcgaggaatt
     3601 ctgcagtcga cggtaccgcg ggcccgggat ccaccggtcg ccaccatggt gagcaagggc
     3661 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     3721 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     3781 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     3841 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     3901 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     3961 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     4021 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     4081 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     4141 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     4201 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     4261 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     4321 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcactcct
     4381 caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa
     4441 taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     4501 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     4561 ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag
     4621 tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg
     4681 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga
     4741 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta
     4801 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta
     4861 ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt
     4921 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4981 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5041 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg
     5101 gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc cagttccgcc
     5161 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     5221 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     5281 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5341 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5401 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5461 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5521 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5581 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5641 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5701 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5761 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5821 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5881 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5941 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6001 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6061 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6121 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6181 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6241 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6301 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6361 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6421 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6481 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6541 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6601 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6661 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6721 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6781 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6841 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6901 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6961 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7021 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7081 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7141 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7201 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7261 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7321 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7381 gcgcacattt ccccgaaaag tgccacctgg gtc
//
