LOCUS       sequence-298088-        4235 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier 1 destination vector for assembling gene cassettes intended for
            expression in bacteria. AB sites. Low copy. Version 1.0..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4235)
  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 4235)
  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).LC_v1
FEATURES             Location/Qualifiers
     source          1..4235
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     180..197
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    212..351
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(277..296)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     437..459
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(453..644)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(691..1773)
                     /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     1735..1754
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1774..1851)
                     /gene="lacI"
                     /label=lacI promoter
     CDS             complement(1978..2283)
                     /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(2080..2099)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2736..3551)
                     /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(2736..3551)
                     /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     3459..3478
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3543..3562
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(3673..4235,1..26)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4162..4181
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
       61 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
      121 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
      181 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
      241 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
      301 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
      361 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
      421 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
      481 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
      541 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
      601 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
      661 gggggatttc tgttcatggg ggtattgcgc tcactgcccg ctttccagtc gggaaacctg
      721 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
      781 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
      841 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
      901 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
      961 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     1021 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     1081 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     1141 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     1201 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     1261 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     1321 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     1381 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     1441 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     1501 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     1561 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     1621 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     1681 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     1741 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     1801 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ctagggataa
     1861 cagggtaatc tacactattg ctgaatcaac cgtgaaaaca acgaagagaa cgctttactt
     1921 acgacactcc gagacagtca gagggtattt attgaactac agcctgcggt ccggtcatta
     1981 tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt ggctgagatc
     2041 agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg tcatcatgcg
     2101 ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt tatctgacag
     2161 cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg ggcgtgtcaa taatatcact
     2221 ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa ccttaaactg
     2281 catttcacca gcccctgttc tcgtcagcaa aagagccgtt catttcaata aaccgggcga
     2341 cctcagccat cccttcctga ttttccgctt tccagcgttc ggcacgcaga cgacgggctt
     2401 cattctgcat ggttgtgctt accagaccgg agatattgac atcatatatg ccttgagcaa
     2461 ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc ttcatagcat acctcttttt
     2521 gacatacttc gggtatacat atcagtatat attcttatac cgcaaaaatc agcgcgcaaa
     2581 tacgcatact gttatctggc ttttagtaag cctcgctggg acgcccgaac ttttacgggt
     2641 gccgactcac tattacagac ttactacaat ctctcagact attggtttca ggctactcaa
     2701 cttccacgaa tcacctttag ggataacagg gtaatttaga aaaactcatc gagcatcaaa
     2761 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2821 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2881 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2941 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     3001 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     3061 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     3121 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     3181 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     3241 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     3301 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     3361 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3421 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3481 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     3541 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3601 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3661 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3721 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3781 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3841 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3901 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3961 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4021 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4081 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4141 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4201 ccacctctga cttgagcgtc gatttttgtg atgct
//
