LOCUS       sequence-275317-        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"
     rep_origin      complement(19..607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(99..118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(781..1572,1573..1641))
                     /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     1404..1423
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1642..1713)
                     /gene="bla"
                     /label=AmpR promoter
     mobile_element  1738..1903
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     enhancer        1997..2300
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2301..2504
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2455..2475
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3006..3025
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      3078..3087
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3090..3113
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3150..3173
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3183..3206
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3225..3248
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(3264..3569)
                     /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(3366..3385)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3933..3956
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3975..3998
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4008..4031
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             4068..4091
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(4186..4206)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4604..4615)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4682..4699)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4688..4895
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        join(4996..5001,5002..5018,5019..5024)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             5213..5746
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     mobile_element  5813..6036
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
ORIGIN
        1 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
       61 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      121 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      181 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
      241 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      301 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      361 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
      421 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
      481 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
      541 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
      601 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
      661 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
      721 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
      781 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
      841 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      901 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      961 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     1021 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     1081 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     1141 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     1201 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     1261 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     1321 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     1381 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     1441 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     1501 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     1561 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     1621 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     1681 ttattgtctc atgagcggat acatatttga atgtatttag gacccaagtc aaccggttgt
     1741 gggcggacaa aatagttggg aactgggagg ggtggaaatg gagtttttaa ggattattta
     1801 gggaagagtg acaaaataga tgggaactgg gtgtagcgtc gtaagctaat acgaaaatta
     1861 aaaatgacaa aatagtttgg aactagattt cacttatctg gttcagcctg cggtccggat
     1921 tcgcccttaa gctagctagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     1981 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     2041 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     2101 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     2161 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     2221 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     2281 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     2341 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     2401 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     2461 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
     2521 agatcctcac tctcttccgc atcgctgtct gcgagggcca gctgttgggc tcgcggttga
     2581 ggacaaactc ttcgcggtct ttccagtact cttggatcgg aaacccgtcg gcctccgaac
     2641 ggtactccgc caccgaggga cctgagcgag tccgcatcga ccggatcgga aaacctctcg
     2701 agaaaggcgt ctaaccagtc acagtcgcaa ggtaggctga gcaccgtggc gggcggcagc
     2761 gggtggcggt cggggttgtt tctggcggag gtgctgctga tgatgtaatt aaagtaggcg
     2821 gtcttgagac ggcggatggt cgaggtgagg tgtggcaggc ttgagatcca gctgttgggg
     2881 tgagtactcc ctctcaaaag cgggcattac ttctgcgcta agattgtcag tttccaaaaa
     2941 cgaggaggat ttgatattca cctggcccga tctggccata cacttgagtg acaatgacat
     3001 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caaagttggc caggcctagt
     3061 tggccaggcc tagtgccgcc accatgggtt ggtcccaccc acaattcgaa aaaggaggtg
     3121 gtgcccgagg cggttcgggt ggaggttcat ggagccatcc gcagtttgaa aagtcatctg
     3181 gtgactataa agacgatgac gataagggcg gtggtagcgg tgggctggaa gttctgttcc
     3241 aggggcccgg atccgaattc tcattatatt ccccagaaca tcaggttaat ggcgtttttg
     3301 atgtcatttt cgcggtggct gagatcagcc acttcttccc cgataacgga gaccggcaca
     3361 ctggccatat cggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa
     3421 agttcacggg agactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt
     3481 cgcccgggcg tgtcaataat atcactctgt acatccacaa acagacgata acggctctct
     3541 cttttatagg tgtaaacctt aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga
     3601 gccgttcatt tcaataaacc gggcgacctc agccatccct tcctgatttt ccgctttcca
     3661 gcgttcggca cgcagacgac gggcttcatt ctgcatggtt gtgcttacca gaccggagat
     3721 attgacatca tatatgcctt gagcaactga tagctgtcgc tgtcaactgt cactgtaata
     3781 cgctgcttca tagcatacct ctttttgaca tacttcgggt atacatatca gtatatattc
     3841 ttataccgca aaaatcagcg cgcaaatacg catactgtta tctggctttt agtaagccga
     3901 gctccgtcga caagcttgcg gccgcactcg agcttgaggt gctttttcaa ggtccgggca
     3961 gcggtggcgg ctctgattac aaggatgacg atgacaaatc atctggttgg agccacccgc
     4021 aattcgagaa aggcggaggc gcacgtggtg ggtcaggagg aggatcttgg tctcatcctc
     4081 agtttgagaa gtgatgataa tggcctgtgg ccgtaatggc ctgtggccgt aaaatcaacc
     4141 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     4201 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     4261 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     4321 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     4381 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     4441 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     4501 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     4561 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     4621 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgagatct
     4681 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4741 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4801 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4861 gaggattggg aagacaatag caggcatgct ggggactcga gttaagggcg aatcgctggg
     4921 acgcccgcga cttcgctgct gcccaaggtt gccgggtgac gcacaccgtg gaaacggatg
     4981 aaggcacgaa cccagttgac ataagcctgt tcggttcgta aactgtaatg caagtagcgt
     5041 atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg
     5101 gcggttttca tggcttgtta tgactgtttt tttgtacagt ctatgcctcg ggcatccaag
     5161 cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg
     5221 cagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttag gtggctcaag
     5281 tatgggcatc attcgcacat gtaggctcgg ccctgaccaa gtcaaatcca tgcgggctgc
     5341 tcttgatctt ttcggtcgtg agttcggaga cgtagccacc tactcccaac atcagccgga
     5401 ctccgattac ctcgggaact tgctccgtag taagacattc atcgcgcttg ctgccttcga
     5461 ccaagaagcg gttgttggcg ctctcgcggc ttacgttctg cccaagtttg agcagccgcg
     5521 tagtgagatc tatatctatg atctcgcagt ctccggcgag caccggaggc agggcattgc
     5581 caccgcgctc atcaatctcc tcaagcatga ggccaacgcg cttggtgctt atgtgatcta
     5641 cgtgcaagca gattacggtg acgatcccgc agtggctctc tatacaaagt tgggcatacg
     5701 ggaagaagtg atgcactttg atatcgaccc aagtaccgcc acctaacaat tcgttcaagc
     5761 cgagatcggc ttcccggccg cggagttgtt cggtaaattg tcacaacgcc gcgaatatag
     5821 tctttacatg cccttggcca cgcccctctt taatacgacg ggcaatttgc acttcagaaa
     5881 atgaagagtt tgctttagcc ataacaaaag tccagtatgc tttttcacag cataactgga
     5941 ctgatttcag tttacaacta ttctgtctag tttaagactt tattgtcata gtttagatct
     6001 attttgttca gtttaagact ttattgtccg cccacacccg cttacgcagg gcatctgcgc
     6061 atccacagga agagcgaccc aagtcaatca ggggataacg caggaaagaa catgtgagca
     6121 aaaggccagc aaaaggccag gaaccgtaaa aa
//
