LOCUS       sequence-137801-        8296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8296)
  AUTHORS   Nie D, Di Nardo A, Han JM, Baharanyi H, Kramvis I, Huynh T, Dabora 
            S, Codeluppi S, Pandolfi PP, Pasquale EB, Sahin M
  TITLE     Tsc2-Rheb signaling regulates EphA-mediated axon guidance.
  JOURNAL   Nat Neurosci. 2010 Feb;13(2):163-72. Epub 2010 Jan 10.
  PUBMED    20062052
REFERENCE   2  (bases 1 to 8296)
  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: "Nat 
            Neurosci."; date: "2010-02"; volume: "13(2)"; pages: "163-72. Epub 
            2010 Jan 10"
COMMENT     Sequence Label: pHAGE-CMV-Rheb(S16H)-IRES-eGFP-W
FEATURES             Location/Qualifiers
     source          1..8296
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        3..306
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        307..510
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     461..481
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     507..531
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     misc_feature    1148..1715
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1165..1717
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1309..1326)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1536..1555
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1729..1731,1732..1734,1735..2448)
                     /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(1729..1731,1732..1734,1735..2445)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1774..1795)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2035..2054)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2382..2403
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2462..3050
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2515..2535)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2933..2944)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3125..3358
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(3477..3495)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3563..3667
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3668..3736,3737..4528)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3886..3905)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4699..5287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5188..5207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5441..5458
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5698..5719
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5734..5739,5740..5757,5758..5764)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5772..5788
                     /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)."
     primer_bind     5777..5799
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5796..5812
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5796..5812
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5862..5881
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             6073..6706
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6753..6878
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7375..7608
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7793..7837
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    8130..8247
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
       61 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      121 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      181 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      241 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      301 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      361 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      421 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      481 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
      541 cgccatccac gctgttttga cctccataga agacaccggc ggccgcacca tgcctcagtc
      601 caagtcccgg aagatcgcca tcctgggcta tcggcatgtg ggaaagtcct cattgacaat
      661 tcagtttgtt gaaggtcaat ttgttgattc ctacgatcca accatagaaa acacattcac
      721 caagctgatc acagtaaatg ggcaagagta tcatcttcag cttgtagaca cagcagggca
      781 ggatgaatat tccatttttc ctcagacata ctccatagat attaatggtt atattcttgt
      841 gtattctgtt acatcaatca aaagctttga agtaattaaa gttatccacg gcaagctgtt
      901 ggacatggtg gggaaagtgc agataccgat tatgttagtc ggaaataaga aggacctgca
      961 tatggaaagg gtgatcagtt atgaagaagg aaaggctttg gcagaatctt ggaatgcagc
     1021 ttttttggaa tcttctgcaa aagaaaatca gactgctgtg gatgttttta gaaggataat
     1081 tttggaagca gaaaagattg acggagcggc ttcacaaggg aagtcttcgt gctcggtgat
     1141 gtgaggatcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt
     1201 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc
     1261 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag
     1321 gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac
     1381 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc
     1441 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc
     1501 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca
     1561 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt
     1621 gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg
     1681 gggacgtggt tttcctttga aaaacacgat gataatatgg ccacacatat ggtgagcaag
     1741 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1801 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1861 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1921 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1981 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2041 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2101 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2161 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     2221 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2281 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     2341 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2401 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaat cgatagatcc
     2461 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2521 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2581 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2641 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2701 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2761 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2821 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     2881 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     2941 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3001 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc ctgagatcct
     3061 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     3121 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
     3181 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     3241 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     3301 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     3361 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     3421 agtgagaggc ccgggttaat taaggaaagg gctagatcat tcttgaagac gaaagggcct
     3481 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     3541 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3601 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3661 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     3721 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3781 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3841 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     3901 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     3961 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4021 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4081 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4141 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4201 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4261 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4321 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4381 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4441 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4501 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4561 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4621 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4681 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4741 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4801 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4861 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4921 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4981 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5041 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5101 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5161 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5221 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5281 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5341 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5401 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5461 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     5521 cagcaagctc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     5581 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     5641 ctccccgtgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5701 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5761 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac
     5821 gaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5881 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa
     5941 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac
     6001 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac
     6061 aaacttagta gttggaaggg ctaattcact cccaaagaag acaagatatc cttgatctgt
     6121 ggatctacca cacacaaggc tacttccctg attagcagaa ctacacacca gggccagggg
     6181 tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag ccagataagg
     6241 tagaagaggc caataaagga gagaacacca gcttgttaca ccctgtgagc ctgcatggga
     6301 tggatgaccc ggagagagaa gtgttagagt ggaggtttga cagccgccta gcatttcatc
     6361 acgtggcccg agagctgcat ccggagtact tcaagaactg ctgatatcga gcttgctaca
     6421 agggactttc cgctggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc
     6481 gagccctcag atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta
     6541 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     6601 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     6661 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     6721 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     6781 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     6841 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     6901 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     6961 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     7021 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     7081 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     7141 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     7201 ccaccgcaca gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga
     7261 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     7321 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     7381 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     7441 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     7501 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     7561 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     7621 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     7681 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     7741 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     7801 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     7861 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     7921 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     7981 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     8041 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgccg
     8101 aattcacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac
     8161 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     8221 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccagtt
     8281 tggactagtg gagttc
//
