LOCUS       sequence-235340-        7091 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 7091)
  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 7091)
  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: "Science. 
            2008 Jun 13. 320(5882):1496-501."
COMMENT     Sequence Label: pLKO.1 GFP shRNA
FEATURES             Location/Qualifiers
     source          1..7091
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..42
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     276..293
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    410..431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(446..451,452..469,470..476)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    484..500
                     /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     489..511
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     508..524
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     508..524
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     543..563
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        545..563
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        591..817
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             818..998
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1045..1170
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1663..1896
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2081..2125
                     /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        2423..2671
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2423..2443
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2594..2613
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2775..2892
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2941..3451
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3370..3388
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3473..4072
                     /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(3473..3492)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3969..3989
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4200..4433
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4505..4626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4542..4561)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4596..4615
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4666..4801
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4821..4840)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4822..4840)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4850..4867)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4850..4866)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4859..4881)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5008..5463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5095..5114)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5305..5326
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5489..5593
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5594..5662,5663..6454)
                     /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(5812..5831)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6625..7091,1..122)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
       61 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      121 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      181 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      241 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
      301 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
      361 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
      421 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
      481 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
      541 gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta
      601 tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg
      661 agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt
      721 aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag
      781 agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat
      841 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
      901 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
      961 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
     1021 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
     1081 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
     1141 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
     1201 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
     1261 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
     1321 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
     1381 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
     1441 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
     1501 cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1561 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1621 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1681 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1741 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1801 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1861 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1921 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1981 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     2041 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     2101 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     2161 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     2221 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     2281 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     2341 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac gagactagcc
     2401 tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat atttgcatat
     2461 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
     2521 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     2581 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     2641 tttatatatc ttgtggaaag gacgaaacac cgggcccgca agctgaccct gaagttcatt
     2701 caagagatga acttcagggt cagcttgctt tttgaattct cgacctcgag acaaatggca
     2761 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     2821 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     2881 attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcc gcggctcgag
     2941 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc
     3001 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc
     3061 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc
     3121 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac
     3181 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc
     3241 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag
     3301 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct
     3361 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct
     3421 cgttgaccga atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccga
     3481 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     3541 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     3601 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     3661 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     3721 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     3781 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     3841 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     3901 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     3961 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     4021 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc
     4081 ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcgg tacctttaag
     4141 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     4201 ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct
     4261 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     4321 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     4381 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag
     4441 ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga
     4501 gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4561 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4621 atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact
     4681 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4741 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4801 ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     4861 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     4921 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     4981 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     5041 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     5101 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5161 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5221 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5281 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5341 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     5401 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     5461 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5521 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5581 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5641 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5701 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5761 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5821 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5881 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5941 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6001 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6061 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6121 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6181 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6241 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6301 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6361 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6421 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6481 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6541 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6601 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6661 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6721 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6781 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6841 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6901 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6961 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7021 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     7081 cggaacagga g
//
