LOCUS       sequence-213989-        8525 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 8525)
  AUTHORS   Thoreen CC, Chantranupong L, Keys HR, Wang T, Gray NS, Sabatini DM
  TITLE     A unifying model for mTORC1-mediated regulation of mRNA translation.
  JOURNAL   Nature. 2012 May 2;485(7396):109-13. doi: 10.1038/nature11083.
  PUBMED    22552098
REFERENCE   2  (bases 1 to 8525)
  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"; doi: "10"; journalName: 
            "Nature"; date: "2012-05-2- 2"; volume: "485"; issue: "7396"; pages:
            "109-13"
COMMENT     Sequence Label: pLJM60-Tia1
FEATURES             Location/Qualifiers
     source          1..8525
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    2..23
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(38..43,44..61,62..68)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    76..92
                     /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     81..103
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     100..116
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     100..116
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     135..155
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        137..155
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        183..409
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             410..590
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    637..762
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1259..1492
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1677..1721
                     /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        2028..2231
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2182..2202
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2228..2252
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2462..2480
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2462..2479
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2493..2512
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             2571..2594
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    3801..3918
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3967..4477
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4396..4414
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4499..5098
                     /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(4499..4518)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4995..5015
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5226..5459
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5531..5652
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5568..5587)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5622..5641
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5692..5827
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5847..5866)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5848..5866)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5876..5893)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5876..5892)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5885..5907)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6034..6489
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6121..6140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6331..6352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6515..6619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6620..6688,6689..7480)
                     /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(6838..6857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7651..8239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8140..8159
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8393..8410
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
       61 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
      121 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt
      181 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
      241 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
      301 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
      361 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
      421 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
      481 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
      541 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
      601 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
      661 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
      721 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
      781 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
      841 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
      901 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
      961 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
     1021 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
     1081 aagaccaccg cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga
     1141 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     1201 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     1261 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     1321 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     1381 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     1441 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     1501 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     1561 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     1621 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     1681 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     1741 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     1801 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     1861 cgtttcagac ccacctccca accccgaggg gaccccatat gccaagtacg ccccctattg
     1921 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1981 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     2041 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     2101 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     2161 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     2221 taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg
     2281 acctccatag aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa
     2341 cgcggattcc ccgtgccaag agtgacgtaa gtaccgccta tagagtctat aggcccaccc
     2401 ccttggcttc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
     2461 gatttaggtg acactataga ataacatcca ctttgccttt ctctccacag gtgtccactc
     2521 ccaggtccaa ctgcacctcg gttctatcga ttgaattcac catggaaatg gactacaagg
     2581 atgacgatga caagggtggt ggtggtggtg cgtcgacgat ggaggacgag atgcccaaga
     2641 ctctatacgt cggtaacctt tctcgagatg tgacagaagc tctcatcctc cagctcttta
     2701 gccagattgg accttgtaaa aactgcaaaa tgattatgga tacagcagga aatgacccat
     2761 actgttttgt ggagttccat gagcaccgtc atgcagctgc agccctggct gccatgaatg
     2821 ggcggaagat aatgggtaag gaagtgaaag tgaattgggc aacaacccct agcagtcaaa
     2881 agaaggatac gagcaatcat ttccatgtgt ttgttggtga cctcagtcca gaaatcacaa
     2941 ccgaagacat caaagcagcg tttgcaccat ttggaagaat ttcagatgcc cgtgtggtaa
     3001 aagacatggc taccgggaag tctaagggat atggctttgt ctcctttttc aacaaatggg
     3061 atgcagaaaa tgccattcag cagatgggtg gccagtggct tggtggaaga caaatcagaa
     3121 ctaactgggc aacccgaaag cctccagctc caaagagtac atatgagtcg aacaccaaac
     3181 agctgtcata tgatgaggtt gtgagtcagt ctagtcccaa caactgcact gtgtactgtg
     3241 gaggggtgac gtcaggactg acagaacaac taatgcgtca gactttttct ccatttggac
     3301 aaataatgga aattcgagtc ttcccagata aaggatactc atttgttcgg ttcagttccc
     3361 atgaaagtgc agcacatgcg attgtttctg ttaatggtac taccattgaa gggcatgtgg
     3421 tgaaatgtta ttggggcaaa gagactcttg atatgataaa ccctgtgcaa cagcaaaatc
     3481 aaattggata tccaccaaca tatggccagt ggggccagtg gtatggaaat gcacaacaga
     3541 ttggccagta tgtgcctaac ggttggcaag tacctgccta tggagtgtat ggccagccat
     3601 ggagccagca ggggttcaat cagacccagt cttctgcacc gtggatggga cccaattaca
     3661 gtgtgccacc acctcaaggg cagaatggca gcatgttgcc tagtcagcct gctgggtatc
     3721 gagtggccgg gtatgaaacc cagtgagcgg ccgcagtgag aattctcgac ctcgagacaa
     3781 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     3841 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     3901 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg
     3961 ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg
     4021 ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt
     4081 cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac
     4141 gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg
     4201 acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat
     4261 ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga
     4321 gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct
     4381 gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg
     4441 ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat
     4501 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     4561 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     4621 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     4681 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     4741 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     4801 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     4861 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     4921 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     4981 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     5041 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg
     5101 cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggtacc
     5161 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     5221 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
     5281 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     5341 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     5401 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     5461 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     5521 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5581 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5641 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
     5701 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     5761 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     5821 ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     5881 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     5941 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     6001 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     6061 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     6121 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     6181 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     6241 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     6301 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     6361 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     6421 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     6481 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     6541 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     6601 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     6661 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6721 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     6781 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     6841 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     6901 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6961 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     7021 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     7081 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     7141 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     7201 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     7261 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7321 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7381 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7441 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     7501 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     7561 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7621 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     7681 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     7741 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     7801 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     7861 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     7921 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7981 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     8041 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     8101 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     8161 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     8221 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     8281 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     8341 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     8401 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     8461 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     8521 cgcaa
//
