LOCUS       sequence-298093-        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      complement(657..1245)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(737..756)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1356..1375)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1367..2182
                     /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             1367..2182
                     /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     complement(1440..1459)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             2635..2940
                     /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     2819..2838
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        3067..3144
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(3145..3742,1..485)
                     /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     complement(3164..3183)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
       61 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
      121 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
      181 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
      241 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
      301 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
      361 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
      421 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
      481 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
      541 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatg
      601 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      661 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      721 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      781 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      841 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      901 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      961 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1021 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1081 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1141 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1201 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1261 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     1321 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     1381 acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg
     1441 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     1501 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     1561 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     1621 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     1681 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     1741 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     1801 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     1861 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     1921 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     1981 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     2041 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     2101 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     2161 gatgctcgat gagtttttct aaattaccct gttatcccta aaggtgattc gtggaagttg
     2221 agtagcctga aaccaatagt ctgagagatt gtagtaagtc tgtaatagtg agtcggcacc
     2281 cgtaaaagtt cgggcgtccc agcgaggctt actaaaagcc agataacagt atgcgtattt
     2341 gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa gtatgtcaaa
     2401 aagaggtatg ctatgaagca gcgtattaca gtgacagttg acagcgacag ctatcagttg
     2461 ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg cagaatgaag
     2521 cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg gctgaggtcg
     2581 cccggtttat tgaaatgaac ggctcttttg ctgacgagaa caggggctgg tgaaatgcag
     2641 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt
     2701 gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
     2761 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
     2821 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
     2881 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa
     2941 tgaccggacc gcaggctgta gttcaataaa taccctctga ctgtctcgga gtgtcgtaag
     3001 taaagcgttc tcttcgttgt tttcacggtt gattcagcaa tagtgtagat taccctgtta
     3061 tccctagaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     3121 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     3181 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     3241 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     3301 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     3361 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     3421 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     3481 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     3541 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     3601 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     3661 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     3721 tcggcgcgtc tgcgtctggc tg
//
