LOCUS       sequence-275322-        3742 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier 1 destination vector for assembling gene cassettes intended for
            expression in bacteria. AB sites. High copy. Version 1.0..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3742)
  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 3742)
  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.assemble(AB).HC_v1
FEATURES             Location/Qualifiers
     source          1..3742
                     /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"
     CDS             complement(837..1919)
                     /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     1881..1900
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1920..1997)
                     /gene="lacI"
                     /label=lacI promoter
     CDS             complement(2124..2429)
                     /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(2226..2245)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2882..3697)
                     /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(2882..3697)
                     /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     3605..3624
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3689..3708
                     /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 tgttcttcta 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 acatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
      781 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
      841 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
      901 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
      961 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     1021 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     1081 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     1141 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     1201 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     1261 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     1321 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     1381 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     1441 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     1501 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     1561 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     1621 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     1681 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     1741 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     1801 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     1861 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     1921 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     1981 cgccattcga tggtgtctag ggataacagg gtaatctaca ctattgctga atcaaccgtg
     2041 aaaacaacga agagaacgct ttacttacga cactccgaga cagtcagagg gtatttattg
     2101 aactacagcc tgcggtccgg tcattatatt ccccagaaca tcaggttaat ggcgtttttg
     2161 atgtcatttt cgcggtggct gagatcagcc acttcttccc cgataacgga gaccggcaca
     2221 ctggccatat cggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa
     2281 agttcacggg agactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt
     2341 cgcccgggcg tgtcaataat atcactctgt acatccacaa acagacgata acggctctct
     2401 cttttatagg tgtaaacctt aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga
     2461 gccgttcatt tcaataaacc gggcgacctc agccatccct tcctgatttt ccgctttcca
     2521 gcgttcggca cgcagacgac gggcttcatt ctgcatggtt gtgcttacca gaccggagat
     2581 attgacatca tatatgcctt gagcaactga tagctgtcgc tgtcaactgt cactgtaata
     2641 cgctgcttca tagcatacct ctttttgaca tacttcgggt atacatatca gtatatattc
     2701 ttataccgca aaaatcagcg cgcaaatacg catactgtta tctggctttt agtaagcctc
     2761 gctgggacgc ccgaactttt acgggtgccg actcactatt acagacttac tacaatctct
     2821 cagactattg gtttcaggct actcaacttc cacgaatcac ctttagggat aacagggtaa
     2881 tttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     2941 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     3001 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     3061 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     3121 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     3181 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     3241 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     3301 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     3361 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     3421 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3481 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3541 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     3601 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3661 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     3721 tgtaagcaga cagttttatt gt
//
