LOCUS       sequence-298070-        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"
     CDS             complement(1..18)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(28..51)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             415..720
                     /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     599..618
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(736..759)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(769..786)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(787..801)
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     promoter        complement(975..1041)
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     promoter        1153..1230
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1231..2313
                     /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(1250..1269)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      complement(2485..3073)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2565..2584)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3184..3203)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3195..4010
                     /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             3195..4010
                     /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(3268..3287)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4026..4044
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 gtgatggtgg tgatggtggc cagagctcgg accttgaaaa agcacctcaa gctcgagtgc
       61 ggccgcaagc ttgtcgacgg agctcggctt actaaaagcc agataacagt atgcgtattt
      121 gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa gtatgtcaaa
      181 aagaggtatg ctatgaagca gcgtattaca gtgacagttg acagcgacag ctatcagttg
      241 ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg cagaatgaag
      301 cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg gctgaggtcg
      361 cccggtttat tgaaatgaac ggctcttttg ctgacgagaa caggggctgg tgaaatgcag
      421 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt
      481 gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
      541 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
      601 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
      661 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa
      721 tgagaattcg gatccgggcc cctggaacag aacttccaga ccagaagaat ggtgatgatg
      781 gtgatgcact ttgtgattca tctagtattc tcctctttaa tctctagagc ctttcggcga
      841 tatggcgtgc tttacagatt ttgaagcgtt aaaggaatat gcactacgag gggtatcaac
      901 gataactctt gaagggactt gccttactac actggatatg cgctagcaca tcaaattgct
      961 atccgctcac aatttgatgt gcattaagcc acgcattggc gggtgatgca acaattattt
     1021 ttcatattta tgattaatcg gccacaccat tccttgcggc ggcggtgctc aacggcctca
     1081 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg
     1141 gaccgcaggc tggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     1201 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     1261 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     1321 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     1381 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     1441 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     1501 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     1561 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     1621 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     1681 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     1741 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     1801 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     1861 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     1921 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     1981 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     2041 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     2101 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     2161 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     2221 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     2281 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     2341 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     2401 cttccggtgg gcgcggggca tgactatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2461 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2521 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2581 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2641 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2701 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2761 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2821 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2881 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     2941 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3001 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3061 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3121 aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta
     3181 atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa
     3241 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     3301 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
     3361 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     3421 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     3481 tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt
     3541 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     3601 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     3661 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     3721 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     3781 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     3841 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     3901 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     3961 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa cgggcgtccc
     4021 agcgagctag ttattgctca gcggtaaaaa aaaccccgcc ctgtcagggg cggggttttt
     4081 ttttggcttt gttagcagcc ggatctcatc a
//
