LOCUS       sequence-275316-        4111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning genes under a cold-shock cspA promoter for 
            expression in bacteria. Version 1.0..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4111)
  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 4111)
  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.cspA_v1
FEATURES             Location/Qualifiers
     source          1..4111
                     /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
     promoter        2109..2175
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     CDS             2349..2363
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     CDS             2364..2381
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2391..2414
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(2430..2735)
                     /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(2532..2551)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3099..3122
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3132..3149
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3217..3235)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3251..4066)
                     /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(3251..4066)
                     /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     3974..3993
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4058..4077
                     /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 accgagatat 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 tggtgtccag cctgcggtcc gggatctcga cgctctccct tatgcgactc
     2041 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     2101 tggtgtggcc gattaatcat aaatatgaaa aataattgtt gcatcacccg ccaatgcgtg
     2161 gcttaatgca catcaaattg tgagcggata gcaatttgat gtgctagcgc atatccagtg
     2221 tagtaaggca agtcccttca agagttatcg ttgatacccc tcgtagtgca tattccttta
     2281 acgcttcaaa atctgtaaag cacgccatat cgccgaaagg ctctagagat taaagaggag
     2341 aatactagat gaatcacaaa gtgcatcacc atcatcacca ttcttctggt ctggaagttc
     2401 tgttccaggg gcccggatcc gaattctcat tatattcccc agaacatcag gttaatggcg
     2461 tttttgatgt cattttcgcg gtggctgaga tcagccactt cttccccgat aacggagacc
     2521 ggcacactgg ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc
     2581 gggtaaagtt cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc
     2641 atccgtcgcc cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg
     2701 ctctctcttt tataggtgta aaccttaaac tgcatttcac cagcccctgt tctcgtcagc
     2761 aaaagagccg ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc
     2821 tttccagcgt tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc
     2881 ggagatattg acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact
     2941 gtaatacgct gcttcatagc atacctcttt ttgacatact tcgggtatac atatcagtat
     3001 atattcttat accgcaaaaa tcagcgcgca aatacgcata ctgttatctg gcttttagta
     3061 agccgagctc cgtcgacaag cttgcggccg cactcgagct tgaggtgctt tttcaaggtc
     3121 cgagctctgg ccaccatcac caccatcact gatgagatcc ggctgctaac aaagccaaaa
     3181 aaaaaccccg cccctgacag ggcggggttt tttttaccgc tgagcaataa ctagctcgct
     3241 gggacgcccg ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     3301 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     3361 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3421 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3481 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     3541 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     3601 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     3661 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     3721 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3781 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3841 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     3901 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     3961 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     4021 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     4081 tactgtttat gtaagcagac agttttattg t
//
