LOCUS       sequence-256358-        5982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses RAP2A in mammalian cells in a G418-selectable plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5982)
  AUTHORS   Walton ZE, Patel CH, Brooks RC, Yu Y, Ibrahim-Hashim A, Riddle M, 
            Porcu A, Jiang T, Ecker BL, Tameire F, Koumenis C, Weeraratna AT, 
            Welsh DK, Gillies R, Alwine JC, Zhang L, Powell JD, Dang CV
  TITLE     Acid Suspends the Circadian Clock in Hypoxia through Inhibition of 
            mTOR.
  JOURNAL   Cell. 2018 Jun 28;174(1):72-87.e32. doi: 10.1016/j.cell.2018.05.009.
            Epub 2018 May 31.
  PUBMED    29861175
REFERENCE   2  (bases 1 to 5982)
  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.cell.2018.05.009"; journalName: "Cell"; date: 
            "2018-06-28- 28"; volume: "174"; issue: "1"; pages: "72-87.e32"
COMMENT     Sequence Label: pcDNA3.1+ Flag-RAP2A
FEATURES             Location/Qualifiers
     source          1..5982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     702..721
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        702..720
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             752..775
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1412..1429)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1418..1642
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1688..2116
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1775..1794)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1985..2006
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2125..2145)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2130..2459
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2310..2445
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2372..2391
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2526..3320
                     /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(2580..2599)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3190..3209
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3494..3615
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3531..3550)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3585..3604
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3664..3680)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3664..3680)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3677..3699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3688..3704
                     /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(3712..3718,3719..3736,3737..3742))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3757..3778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3895..3912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4066..4654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4146..4165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4825..5616,5617..5685))
                     /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     5448..5467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5686..5790)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5865..5884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      661 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
      721 gagacccaag ctggctagcg ctaccggtat ggactacaag gatgacgatg acaagggtgg
      781 tggtggtggt gcgtcgacga tgcgcgagta caaagtggtg gtgctgggct cgggcggggt
      841 aggcaaatcc gccctgaccg tgcagttcgt gaccggcacc ttcatcgaga aatacgaccc
      901 caccatcgag gacttctacc gcaaggagat cgaggtggat tcgtcgccgt cggtgctgga
      961 gatcctggac acggcgggca ccgagcagtt cgcgtccatg cgggacctgt acatcaagaa
     1021 cggccagggc ttcatcctcg tctacagcct cgtcaaccag cagagcttcc aggacatcaa
     1081 gcccatgcgg gaccagatca tccgcgtgaa gcggtatgag aaagtgccag tcatcttggt
     1141 tgggaacaaa gtggacctgg aaagtgagag agaagtatcg tccagcgaag gcagagccct
     1201 tgctgaagag tggggctgcc cctttatgga aacttccgct aagagtaaaa caatggtgga
     1261 cgaactcttt gcagaaattg tgaggcagat gaactatgct gctcagcctg acaaagatga
     1321 cccatgctgt tctgcatgta acatacaata ggatatccag cacagtggcg gccgctcgag
     1381 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     1441 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     1501 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     1561 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     1621 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     1681 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1741 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1801 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1861 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1921 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1981 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2041 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2101 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     2161 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     2221 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2281 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     2341 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     2401 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     2461 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     2521 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     2581 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     2641 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     2701 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     2761 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     2821 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     2881 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     2941 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     3001 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     3061 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     3121 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     3181 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     3241 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     3301 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     3361 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     3421 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     3481 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3541 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3601 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     3661 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3721 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3781 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3841 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3901 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3961 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4021 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4081 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4141 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4201 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4261 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4321 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4381 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4441 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4501 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4561 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4621 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4681 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4741 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4801 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4861 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4921 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4981 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5041 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5101 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5161 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5221 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5281 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5341 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5401 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5461 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5521 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5581 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5641 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5701 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5761 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     5821 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
     5881 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
     5941 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg ac
//
