LOCUS       sequence-298068-        4349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning genes under a T7 promoter for expression in
            bacteria. Version 1.0..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4349)
  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 4349)
  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: pFloat.T7_v1
FEATURES             Location/Qualifiers
     source          1..4349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..78)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     98..117
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        98..116
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    122..138
                     /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)."
     CDS             190..207
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             217..240
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(256..561)
                     /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(358..377)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             925..948
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             958..975
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1043..1061)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1077..1892)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1077..1892)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1800..1819
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1884..1903
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2014..2602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2503..2522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2756..2773
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2788..2927
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2853..2872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3013..3035
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3029..3220)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3267..4349)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4311..4330
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
       61 gcgccattcg atggtgtcca gcctgcggtc cggaaattaa tacgactcac tatagggaga
      121 attgtgagcg gataacaatt tcacacaaga aataattttt ctagagatta aagaggagaa
      181 tactagatgc atcaccatca tcaccattct tctggtctgg aagttctgtt ccaggggccc
      241 ggatccgaat tctcattata ttccccagaa catcaggtta atggcgtttt tgatgtcatt
      301 ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gagaccggca cactggccat
      361 atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt aaagttcacg
      421 ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc gtcgcccggg
      481 cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct ctcttttata
      541 ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa gagccgttca
      601 tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc cagcgttcgg
      661 cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag atattgacat
      721 catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa tacgctgctt
      781 catagcatac ctctttttga catacttcgg gtatacatat cagtatatat tcttataccg
      841 caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc gagctccgtc
      901 gacaagcttg cggccgcact cgagcttgag gtgctttttc aaggtccgag ctctggccac
      961 catcaccacc atcactgatg agatccggct gctaacaaag ccaaaaaaaa accccgcccc
     1021 tgacagggcg gggttttttt taccgctgag caataactag ctcgctggga cgcccgttag
     1081 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1141 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     1201 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1261 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1321 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     1381 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     1441 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     1501 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1561 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     1621 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     1681 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     1741 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     1801 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     1861 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     1921 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1981 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2041 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2101 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2161 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2221 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2281 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2341 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2401 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2461 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2521 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2581 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2641 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     2701 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2761 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     2821 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2881 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     2941 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3001 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3061 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     3121 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     3181 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     3241 gatttctgtt catgggggta ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3301 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     3361 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     3421 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     3481 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     3541 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     3601 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     3661 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     3721 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     3781 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     3841 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     3901 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     3961 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4021 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4081 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4141 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     4201 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     4261 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     4321 gcgacatcgt ataacgttac tggtttcac
//
