LOCUS       Exported                8431 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Flag pLJM1 RagB wt
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8431)
  AUTHORS   Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled
            L, Sabatini DM
  TITLE     The Rag GTPases bind raptor and mediate amino acid signaling to 
            mTORC1.
  JOURNAL   Science. 2008 Jun 13. 320(5882):1496-501.
  PUBMED    18497260
REFERENCE   2  (bases 1 to 8431)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8431
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        131..334
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     285..305
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             375..398
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1514..1631
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1680..2190
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2109..2127
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2212..2811
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2212..2231)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2708..2728
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2939..3172
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3244..3365
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3281..3300)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3335..3354
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3405..3540
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3467..3486
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(3560..3579)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3561..3579)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3589..3606)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3589..3605)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3598..3620)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3747..4202
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3834..3853)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4044..4065
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4228..4332
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4333..5193
                     /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(4551..4570)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5364..5952
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5853..5872
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6106..6123
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6240..6261
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6276..6306
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6314..6330
                     /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     6319..6341
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6338..6354
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6338..6354
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6373..6393
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6375..6393
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6421..6647
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6648..6828
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6875..7000
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7493..7726
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7911..7955
                     /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"
     primer_bind     8253..8273
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
       61 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      121 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      181 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      241 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      301 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      361 gcgctaccgg tatggactac aaggatgacg atgacaaggg tggtggtggt ggtgcgtcga
      421 cgatggaaga atctgactct gagaaaacga cggagaaaga aaatctgggg ccgagaatgg
      481 atccaccact aggggaaccg gaaggatcgc ttgggtgggt gctaccaaat acagccatga
      541 agaaaaaggt gctgttgatg ggtaaaagtg ggtctggtaa gaccagcatg aggtctatta
      601 tctttgcaaa ttatattgcc agagacacac gtcgccttgg cgcaacaatt gatgtagaac
      661 attctcatgt tcgatttctg ggaaacctgg tattgaacct gtgggattgt ggtgggcaag
      721 acaccttcat ggaaaattat ttcactagcc aacgggacaa catcttccga aatgtggagg
      781 ttctgattta tgtctttgat gtggagagcc gcgaactgga aaaggacatg cactattacc
      841 aatcatgcct ggaggccatt ctgcagaatt ctccagatgc caaaatattt tgcttggtac
      901 acaaaatgga tctggtacag gaggatcaac gggacctgat ttttaaagag cgagaagaag
      961 atttgaggcg tttgtctcgc ccattggaat gttcttgttt ccgaacatct atctgggatg
     1021 aaaccctcta taaggcttgg tccagcatag tttatcagct gattcccaat gttcagcagc
     1081 tggaaatgaa cctaaggaat tttgctgaaa ttatcgaagc tgatgaagta cttctttttg
     1141 agagagctac ttttctggta atttctcact atcagtgtaa agagcagcgt gatgcccata
     1201 gatttgagaa aataagcaac attattaagc agttcaagct gagctgcagc aagctggctg
     1261 cctctttcca gagtatggaa gtcaggaact ctaacttcgc tgctttcatt gacatcttta
     1321 catccaacac ttatgtgatg gttgtgatgt ctgatccgtc cattccttct gcagctactc
     1381 tgatcaacat ccgcaatgcc aggaaacact ttgaaaagct ggaaagagtg gatggaccaa
     1441 agcagtgtct tctcatgcgc taagatatct tcgaattctc gacctcgaga caaatggcag
     1501 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     1561 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     1621 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg
     1681 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct
     1741 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc
     1801 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct
     1861 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg
     1921 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg
     1981 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc
     2041 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg
     2101 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc
     2161 gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag
     2221 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     2281 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     2341 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     2401 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     2461 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     2521 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     2581 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     2641 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     2701 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     2761 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc
     2821 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga
     2881 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     2941 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     3001 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     3061 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     3121 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt
     3181 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     3241 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3301 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3361 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     3421 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3481 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3541 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     3601 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     3661 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     3721 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     3781 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3841 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3901 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3961 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4021 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4081 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4141 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     4201 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4261 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4321 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4381 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4441 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4501 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4561 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4621 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4681 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4741 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4801 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4861 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4921 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4981 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5041 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5101 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5161 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5221 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5281 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5341 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5401 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5461 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5521 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5581 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5641 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5701 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5761 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5821 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5881 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5941 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6001 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6061 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6121 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6181 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6241 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     6301 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     6361 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
     6421 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
     6481 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
     6541 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
     6601 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
     6661 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     6721 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     6781 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
     6841 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
     6901 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
     6961 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
     7021 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
     7081 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
     7141 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
     7201 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
     7261 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
     7321 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     7381 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     7441 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     7501 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     7561 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     7621 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     7681 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     7741 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     7801 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     7861 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     7921 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     7981 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     8041 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     8101 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     8161 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac
     8221 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat
     8281 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     8341 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     8401 ttttaaaatt atgttttaaa atggactatc a
//
