LOCUS       sequence-214705-        6007 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6007)
  AUTHORS   Urano J, Sato T, Matsuo T, Otsubo Y, Yamamoto M, Tamanoi F
  TITLE     Point mutations in TOR confer Rheb-independent growth in fission 
            yeast and nutrient-independent mammalian TOR signaling in mammalian 
            cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2007 Feb 27. 104(9):3514-9.
  PUBMED    17360675
REFERENCE   2  (bases 1 to 6007)
  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"; journalName: "Proc Natl 
            Acad Sci U S A. 2007 Feb 27. 104(9):3514-9."
COMMENT     Sequence Label: pcDNA3-FLAG-Rheb
FEATURES             Location/Qualifiers
     source          1..6007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             950..973
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1560..1578)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1561..1578)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1598..1615)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1604..1828
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1874..2302
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1961..1980)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2171..2192
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2311..2331)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2316..2645
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2496..2631
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2558..2577
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2712..3506
                     /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(2766..2785)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3376..3395
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3680..3801
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3717..3736)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3771..3790
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3850..3866)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3850..3866)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3863..3885)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3874..3890
                     /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(3898..3904,3905..3922,3923..3928))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3943..3964
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4081..4098)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4252..4840)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4332..4351)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5011..5802,5803..5871))
                     /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     5634..5653
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5872..5976)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcaccatgg attacaagga
      961 tgacgatgac aagccgcagt ccaagtcccg gaagatcgcg atcctgggct accggtctgt
     1021 ggggaaatcc tcattgacga ttcaatttgt tgaaggccaa tttgtggact cctacgatcc
     1081 aaccatagaa aacactttta caaagttgat cacagtaaat ggacaagaat atcatcttca
     1141 acttgtagac acagccgggc aagatgaata ttctatcttt cctcagacat actccataga
     1201 tattaatggc tatattcttg tgtattctgt tacatcaatc aaaagttttg aagtgattaa
     1261 agttatccat ggcaaattgt tggatatggt ggggaaagta caaataccta ttatgttggt
     1321 tgggaataag aaagacctgc atatggaaag ggtgatcagt tatgaagaag ggaaagcttt
     1381 ggcagaatct tggaatgcag cttttttgga atcttctgct aaagaaaatc agactgctgt
     1441 ggatgttttt cgaaggataa ttttggaggc agaaaaaatg gacggggcag cttcacaagg
     1501 caagtcttca tgctcggtga tgtgagcggc cgctcgagca tgcatctaga gggccctatt
     1561 ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     1621 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1681 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     1741 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     1801 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     1861 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1921 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1981 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2041 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2101 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2161 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2221 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2281 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     2341 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2401 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2461 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     2521 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     2581 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     2641 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     2701 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     2761 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     2821 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     2881 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     2941 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3001 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3061 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3121 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3181 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     3241 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     3301 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     3361 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     3421 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     3481 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     3541 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     3601 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3661 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3721 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3781 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     3841 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3901 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3961 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4021 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4081 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4141 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4201 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4261 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4321 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4381 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4441 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4501 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4561 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4621 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4681 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4741 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4801 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4861 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4921 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4981 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5041 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5101 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5161 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5221 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5281 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5341 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5401 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5461 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5521 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5581 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5641 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5701 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5761 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5821 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5881 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5941 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6001 tgacgtc
//
