LOCUS       sequence-298087-        6152 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning genes under an enhanced CMVT promoter for 
            expression in mammalian cells. Version 1.0..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6152)
  AUTHORS   Rogala KB, Gu X, Kedir JF, Abu-Remaileh M, Bianchi LF, Bottino AMS, 
            Dueholm R, Niehaus A, Overwijn D, Fils AP, Zhou SX, Leary D, Laqtom 
            NN, Brignole EJ, Sabatini DM
  TITLE     Structural basis for the docking of mTORC1 on the lysosomal surface.
  JOURNAL   Science. 2019 Oct 25;366(6464):468-475. doi: 
            10.1126/science.aay0166. Epub 2019 Oct 10.
  PUBMED    31601708
REFERENCE   2  (bases 1 to 6152)
  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: 
            "10.1126/science.aay0166"; journalName: "Science"; date: 
            "2019-10-25- 25"; volume: "366"; issue: "6464"; pages: "468-475"
COMMENT     Sequence Label: pDarmo.CMVT_v1
FEATURES             Location/Qualifiers
     source          1..6152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     mobile_element  1..166
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     enhancer        260..563
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        564..767
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     718..738
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1269..1288
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1341..1350
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1353..1376
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1413..1436
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1446..1469
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1488..1511
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1527..1832)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1629..1648)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2196..2219
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2238..2261
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2271..2294
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2331..2354
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(2449..2469)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2867..2878)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2945..2962)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2951..3158
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        join(3259..3264,3265..3281,3282..3287)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3476..4009
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     mobile_element  4076..4299
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     rep_origin      complement(4434..5022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4514..4533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5196..5987,5988..6056))
                     /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     5819..5838
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6057..6128)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgtgggcgga caaaatagtt gggaactggg aggggtggaa atggagtttt taaggattat
       61 ttagggaaga gtgacaaaat agatgggaac tgggtgtagc gtcgtaagct aatacgaaaa
      121 ttaaaaatga caaaatagtt tggaactaga tttcacttat ctggttcagc ctgcggtccg
      181 gattcgccct taagctagct agttattaat agtaatcaat tacggggtca ttagttcata
      241 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      301 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      361 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      421 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      481 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      541 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      601 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      661 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      721 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
      781 gtcagatcct cactctcttc cgcatcgctg tctgcgaggg ccagctgttg ggctcgcggt
      841 tgaggacaaa ctcttcgcgg tctttccagt actcttggat cggaaacccg tcggcctccg
      901 aacggtactc cgccaccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacctc
      961 tcgagaaagg cgtctaacca gtcacagtcg caaggtaggc tgagcaccgt ggcgggcggc
     1021 agcgggtggc ggtcggggtt gtttctggcg gaggtgctgc tgatgatgta attaaagtag
     1081 gcggtcttga gacggcggat ggtcgaggtg aggtgtggca ggcttgagat ccagctgttg
     1141 gggtgagtac tccctctcaa aagcgggcat tacttctgcg ctaagattgt cagtttccaa
     1201 aaacgaggag gatttgatat tcacctggcc cgatctggcc atacacttga gtgacaatga
     1261 catccacttt gcctttctct ccacaggtgt ccactcccag gtccaaagtt ggccaggcct
     1321 agttggccag gcctagtgcc gccaccatgg gttggtccca cccacaattc gaaaaaggag
     1381 gtggtgcccg aggcggttcg ggtggaggtt catggagcca tccgcagttt gaaaagtcat
     1441 ctggtgacta taaagacgat gacgataagg gcggtggtag cggtgggctg gaagttctgt
     1501 tccaggggcc cggatccgaa ttctcattat attccccaga acatcaggtt aatggcgttt
     1561 ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac ggagaccggc
     1621 acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg caccaccggg
     1681 taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg gatcaccatc
     1741 cgtcgcccgg gcgtgtcaat aatatcactc tgtacatcca caaacagacg ataacggctc
     1801 tctcttttat aggtgtaaac cttaaactgc atttcaccag cccctgttct cgtcagcaaa
     1861 agagccgttc atttcaataa accgggcgac ctcagccatc ccttcctgat tttccgcttt
     1921 ccagcgttcg gcacgcagac gacgggcttc attctgcatg gttgtgctta ccagaccgga
     1981 gatattgaca tcatatatgc cttgagcaac tgatagctgt cgctgtcaac tgtcactgta
     2041 atacgctgct tcatagcata cctctttttg acatacttcg ggtatacata tcagtatata
     2101 ttcttatacc gcaaaaatca gcgcgcaaat acgcatactg ttatctggct tttagtaagc
     2161 cgagctccgt cgacaagctt gcggccgcac tcgagcttga ggtgcttttt caaggtccgg
     2221 gcagcggtgg cggctctgat tacaaggatg acgatgacaa atcatctggt tggagccacc
     2281 cgcaattcga gaaaggcgga ggcgcacgtg gtgggtcagg aggaggatct tggtctcatc
     2341 ctcagtttga gaagtgatga taatggcctg tggccgtaat ggcctgtggc cgtaaaatca
     2401 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     2461 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     2521 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     2581 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     2641 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     2701 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     2761 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     2821 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     2881 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgaga
     2941 tctgcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     3001 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     3061 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     3121 ggggaggatt gggaagacaa tagcaggcat gctggggact cgagttaagg gcgaatcgct
     3181 gggacgcccg cgacttcgct gctgcccaag gttgccgggt gacgcacacc gtggaaacgg
     3241 atgaaggcac gaacccagtt gacataagcc tgttcggttc gtaaactgta atgcaagtag
     3301 cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca
     3361 gtggcggttt tcatggcttg ttatgactgt ttttttgtac agtctatgcc tcgggcatcc
     3421 aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt
     3481 acgcagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taggtggctc
     3541 aagtatgggc atcattcgca catgtaggct cggccctgac caagtcaaat ccatgcgggc
     3601 tgctcttgat cttttcggtc gtgagttcgg agacgtagcc acctactccc aacatcagcc
     3661 ggactccgat tacctcggga acttgctccg tagtaagaca ttcatcgcgc ttgctgcctt
     3721 cgaccaagaa gcggttgttg gcgctctcgc ggcttacgtt ctgcccaagt ttgagcagcc
     3781 gcgtagtgag atctatatct atgatctcgc agtctccggc gagcaccgga ggcagggcat
     3841 tgccaccgcg ctcatcaatc tcctcaagca tgaggccaac gcgcttggtg cttatgtgat
     3901 ctacgtgcaa gcagattacg gtgacgatcc cgcagtggct ctctatacaa agttgggcat
     3961 acgggaagaa gtgatgcact ttgatatcga cccaagtacc gccacctaac aattcgttca
     4021 agccgagatc ggcttcccgg ccgcggagtt gttcggtaaa ttgtcacaac gccgcgaata
     4081 tagtctttac atgcccttgg ccacgcccct ctttaatacg acgggcaatt tgcacttcag
     4141 aaaatgaaga gtttgcttta gccataacaa aagtccagta tgctttttca cagcataact
     4201 ggactgattt cagtttacaa ctattctgtc tagtttaaga ctttattgtc atagtttaga
     4261 tctattttgt tcagtttaag actttattgt ccgcccacac ccgcttacgc agggcatctg
     4321 cgcatccaca ggaagagcga cccaagtcaa tcaggggata acgcaggaaa gaacatgtga
     4381 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4441 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4501 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4561 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4621 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4681 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4741 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4801 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4861 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4921 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4981 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5041 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5101 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5161 taaagtatat atgagtaaac ttggtctgac agttattacc aatgcttaat cagtgaggca
     5221 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5281 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5341 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5401 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5461 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5521 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5581 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5641 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5701 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5761 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5821 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5881 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5941 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6001 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6061 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6121 tttgaatgta tttaggaccc aagtcaaccg gt
//
