LOCUS       sequence-275535-        4238 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 4238)
  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 4238)
  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..4238
                     /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..993
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(919..938)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1079..1101
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1095..1286)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1333..2415)
                     /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     2377..2396
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2416..2493)
                     /gene="lacI"
                     /label=lacI promoter
     CDS             complement(2620..2925)
                     /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(2722..2741)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3378..4193)
                     /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(3378..4193)
                     /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     4101..4120
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4185..4204
                     /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 tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
      961 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     1021 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     1081 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     1141 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     1201 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     1261 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     1321 ggggtattgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     1381 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     1441 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     1501 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     1561 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     1621 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     1681 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     1741 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     1801 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     1861 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     1921 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     1981 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     2041 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     2101 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     2161 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     2221 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     2281 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     2341 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     2401 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     2461 accgcgaaag gttttgcgcc attcgatggt gtctagggat aacagggtaa tctacactat
     2521 tgctgaatca accgtgaaaa caacgaagag aacgctttac ttacgacact ccgagacagt
     2581 cagagggtat ttattgaact acagcctgcg gtccggtcat tatattcccc agaacatcag
     2641 gttaatggcg tttttgatgt cattttcgcg gtggctgaga tcagccactt cttccccgat
     2701 aacggagacc ggcacactgg ccatatcggt ggtcatcatg cgccagcttt catccccgat
     2761 atgcaccacc gggtaaagtt cacgggagac tttatctgac agcagacgtg cactggccag
     2821 ggggatcacc atccgtcgcc cgggcgtgtc aataatatca ctctgtacat ccacaaacag
     2881 acgataacgg ctctctcttt tataggtgta aaccttaaac tgcatttcac cagcccctgt
     2941 tctcgtcagc aaaagagccg ttcatttcaa taaaccgggc gacctcagcc atcccttcct
     3001 gattttccgc tttccagcgt tcggcacgca gacgacgggc ttcattctgc atggttgtgc
     3061 ttaccagacc ggagatattg acatcatata tgccttgagc aactgatagc tgtcgctgtc
     3121 aactgtcact gtaatacgct gcttcatagc atacctcttt ttgacatact tcgggtatac
     3181 atatcagtat atattcttat accgcaaaaa tcagcgcgca aatacgcata ctgttatctg
     3241 gcttttagta agcctcgctg ggacgcccga acttttacgg gtgccgactc actattacag
     3301 acttactaca atctctcaga ctattggttt caggctactc aacttccacg aatcaccttt
     3361 agggataaca gggtaattta gaaaaactca tcgagcatca aatgaaactg caatttattc
     3421 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     3481 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     3541 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     3601 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     3661 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     3721 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     3781 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     3841 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     3901 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     3961 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     4021 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     4081 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     4141 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     4201 cttgtattac tgtttatgta agcagacagt tttattgt
//
