LOCUS       sequence-153817-        7084 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 7084)
  AUTHORS   Thoreen CC, Kang SA, Chang JW, Liu Q, Zhang J, Gao Y, Reichling LJ, 
            Sim T, Sabatini DM, Gray NS
  TITLE     An ATP-competitive mammalian target of rapamycin inhibitor reveals 
            rapamycin-resistant functions of mTORC1.
  JOURNAL   J Biol Chem. 2009 Mar 20. 284(12):8023-32.
  PUBMED    19150980
REFERENCE   2  (bases 1 to 7084)
  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: "J Biol 
            Chem. 2009 Mar 20. 284(12):8023-32."
COMMENT     Sequence Label: pLKO mouse shRNA 1 rictor
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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        join(91..96,97..114,115..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..2316
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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    2413..2530
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2579..3089
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3008..3026
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3111..3710
                     /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(3111..3130)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3607..3627
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3838..4071
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4143..4264
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4180..4199)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4234..4253
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4304..4439
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4459..4478)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4460..4478)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4488..4505)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4488..4504)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4497..4519)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4646..5101
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4733..4752)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4943..4964
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5127..5231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5232..5300,5301..6092)
                     /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(5450..5469)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6263..6851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6752..6771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7005..7022
                     /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 aacaccgggc cagtaagatg ggaatcattc
     2341 tcgagaatga ttcccatctt actggctttt tgaattctcg acctcgagac aaatggcagt
     2401 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2461 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2521 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggccgc ggctcgaggg
     2581 ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg
     2641 ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg
     2701 ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg
     2761 ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg
     2821 aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc
     2881 gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg
     2941 gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc
     3001 ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg
     3061 ttgaccgaat caccgacctc tctccccagg gggatccacc ggagcttacc atgaccgagt
     3121 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     3181 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     3241 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3301 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     3361 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     3421 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     3481 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     3541 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     3601 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3661 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc
     3721 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcggta cctttaagac
     3781 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     3841 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     3901 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     3961 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     4021 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt
     4081 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     4141 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4201 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4261 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
     4321 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4381 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4441 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     4501 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4561 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4621 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4681 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4741 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4801 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4861 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4921 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4981 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5041 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     5101 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5161 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5221 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5281 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5341 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5401 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5461 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5521 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5581 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5641 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5701 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5761 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5821 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5881 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5941 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6001 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6061 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6121 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6181 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6241 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6301 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6361 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6421 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6481 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6541 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6601 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6661 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6721 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6781 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6841 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6901 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6961 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     7021 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     7081 aatg
//
