LOCUS       Exported                7088 bp ds-DNA     circular SYN 01-SEP-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    mTOR_2 shRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7088)
  AUTHORS   Sarbassov DD, Guertin DA, Ali SM, Sabatini DM
  TITLE     Phosphorylation and regulation of Akt/PKB by the rictor-mTOR 
            complex.
  JOURNAL   Science 2005 Feb 18;307(5712):1098-101.
  PUBMED    15718470
REFERENCE   2  (bases 1 to 7088)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 1, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7088
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    55..76
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        91..121
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    129..145
                     /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     134..156
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     153..169
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     153..169
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     188..208
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        190..208
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        236..462
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             463..643
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    690..815
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1308..1541
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1726..1770
                     /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"
     promoter        2068..2308
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2068..2088
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2239..2258
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2417..2534
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2583..3093
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3012..3030
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3115..3714
                     /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(3115..3134)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3611..3631
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3842..4075
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4147..4268
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4184..4203)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4238..4257
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4308..4443
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4370..4389
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4463..4482)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4464..4482)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4492..4509)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4492..4508)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4501..4523)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4650..5105
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4737..4756)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4947..4968
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5131..5235
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5236..6096
                     /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(5454..5473)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6267..6855
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6756..6775
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7009..7026
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
       61 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      121 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      181 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag
      241 tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta
      301 caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc
      361 ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat
      421 tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac
      481 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
      541 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
      601 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
      661 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
      721 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
      781 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
      841 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
      901 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
      961 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     1021 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     1081 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     1141 ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1201 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1261 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1321 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1381 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1441 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1501 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1561 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     1621 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     1681 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     1741 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     1801 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     1861 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     1921 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     1981 agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg atcacgagac
     2041 tagcctcgag cggccgcccc cttcaccgag ggcctatttc ccatgattcc ttcatatttg
     2101 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     2161 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2221 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2281 ttggctttat atatcttgtg gaaaggacga aacaccggcc gcattgtctc tatcaagttc
     2341 ttcctgtcaa acttgataga gacaatgcgg tttttgaatt ctcgacctcg agacaaatgg
     2401 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     2461 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     2521 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg ccgcggctcg
     2581 agggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc
     2641 tctgggcgtg gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg
     2701 tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt
     2761 cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa
     2821 acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca
     2881 gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc
     2941 agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc
     3001 ctgcccgcgc ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc
     3061 ctcgttgacc gaatcaccga cctctctccc cagggggatc caccggagct taccatgacc
     3121 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     3181 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     3241 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     3301 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     3361 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     3421 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     3481 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     3541 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     3601 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     3661 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg
     3721 ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc ggtaccttta
     3781 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     3841 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     3901 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     3961 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     4021 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt
     4081 agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt
     4141 gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4201 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4261 gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa
     4321 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4381 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4441 gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
     4501 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     4561 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     4621 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     4681 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4741 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4801 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4861 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4921 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4981 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5041 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5101 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5161 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5221 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5281 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5341 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5401 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5461 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5521 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5581 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5641 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5701 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5761 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5821 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5881 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5941 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6001 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6061 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6121 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6181 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6241 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6301 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6361 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6421 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6481 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6541 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6601 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6661 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6721 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6781 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6841 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6901 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6961 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     7021 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     7081 cattaatg
//
