LOCUS       sequence-275315-        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"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     819..836
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    851..990
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(916..935)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1076..1098
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1092..1283)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1330..2412)
                     /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     2374..2393
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2413..2490)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2510..2529
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2510..2528
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2534..2550
                     /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             2602..2619
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2629..2652
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(2668..2973)
                     /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(2770..2789)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3337..3360
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3370..3387
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3455..3473)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3489..4304)
                     /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(3489..4304)
                     /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     4212..4231
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4296..4315
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      841 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      901 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      961 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     1021 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     1081 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     1141 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     1201 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     1261 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     1321 gtattgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1381 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     1441 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     1501 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     1561 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     1621 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     1681 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     1741 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     1801 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     1861 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     1921 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     1981 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     2041 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     2101 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     2161 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     2221 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     2281 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     2341 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     2401 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     2461 gcgaaaggtt ttgcgccatt cgatggtgtc cagcctgcgg tccggaaatt aatacgactc
     2521 actataggga gaattgtgag cggataacaa tttcacacaa gaaataattt ttctagagat
     2581 taaagaggag aatactagat gcatcaccat catcaccatt cttctggtct ggaagttctg
     2641 ttccaggggc ccggatccga attctcatta tattccccag aacatcaggt taatggcgtt
     2701 tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa cggagaccgg
     2761 cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat gcaccaccgg
     2821 gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg ggatcaccat
     2881 ccgtcgcccg ggcgtgtcaa taatatcact ctgtacatcc acaaacagac gataacggct
     2941 ctctctttta taggtgtaaa ccttaaactg catttcacca gcccctgttc tcgtcagcaa
     3001 aagagccgtt catttcaata aaccgggcga cctcagccat cccttcctga ttttccgctt
     3061 tccagcgttc ggcacgcaga cgacgggctt cattctgcat ggttgtgctt accagaccgg
     3121 agatattgac atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt
     3181 aatacgctgc ttcatagcat acctcttttt gacatacttc gggtatacat atcagtatat
     3241 attcttatac cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc ttttagtaag
     3301 ccgagctccg tcgacaagct tgcggccgca ctcgagcttg aggtgctttt tcaaggtccg
     3361 agctctggcc accatcacca ccatcactga tgagatccgg ctgctaacaa agccaaaaaa
     3421 aaaccccgcc cctgacaggg cggggttttt tttaccgctg agcaataact agctcgctgg
     3481 gacgcccgtt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     3541 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     3601 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     3661 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     3721 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     3781 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     3841 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     3901 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     3961 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     4021 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     4081 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     4141 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     4201 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     4261 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     4321 ctgtttatgt aagcagacag ttttattgt
//
