LOCUS       sequence-273231-        4355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of p14 and MP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4355)
  AUTHORS   Shen K, Sabatini DM
  TITLE     Ragulator and SLC38A9 activate the Rag GTPases through noncanonical 
            GEF mechanisms.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Sep 18;115(38):9545-9550. doi: 
            10.1073/pnas.1811727115. Epub 2018 Sep 4.
  PUBMED    30181260
REFERENCE   2  (bases 1 to 4355)
  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: 
            "10.1073/pnas.1811727115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-09-18- 18"; volume: "115"; issue: "38"; pages: 
            "9545-9550"
COMMENT     Sequence Label: [pSK131] pCOLADuet p14|MP1
FEATURES             Location/Qualifiers
     source          1..4355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     promoter        520..538
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    539..563
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     CDS             1002..1046
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1084..1102)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1098..1145
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1375..2190)
                     /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(1375..2190)
                     /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     2098..2117
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2191..2282)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2300..2935)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(2445..2464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3133..4215)
                     /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     4177..4196
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4216..4293)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4339..4355,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggaatgc tgcgtcccaa ggctttgacg caggtgctaa gccaagccaa
      121 cactggagga gtccagagca ccctgctgct gaataatgag ggatcgctgc tggcctactc
      181 cggttatggg gacacagatg cccgggtcac tgcggccatc gccagtaaca tctgggccgc
      241 gtatgatagg aacgggaacc aagcgtttaa tgaagacagt ctcaaattta tcctgatgga
      301 ctgcatggag ggccgtgtag ccattacgag ggtggccaac cttctgctat gtatgtatgc
      361 caaggagacc gtaggcttcg gaatgctcaa ggctaaggcc caggccctgg tgcagtacct
      421 ggaggaaccc ctcacccaag tagcagcatc ataagcggcc gcataatgct taagtcgaac
      481 agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc actatagggg
      541 aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa gaaggagata
      601 tacatatggc ggatgatcta aagcgattcc tgtacaaaaa gttgccaagt gttgaagggc
      661 tccatgcaat tgttgtgtca gatagagacg gggtgccggt tattaaagtg gctaacgaca
      721 gtgccccaga gcacgccctg agacctggct tcctatccac gtttgccctc gcaacagacc
      781 aaggcagcaa actcggactt tcaaaaaaca aaagcatcat ctgctactat aacacctacc
      841 aggtggttca gttcaatcgt ttgcctctgg tggtgagttt catagccagc agcagtgcca
      901 acacaggact aattgtcagc ctagaaaagg agctagctcc attatttgaa gaactgataa
      961 aagttgtgga agtgtctact agtagttgac tcgagtctgg taaagaaacc gctgctgcga
     1021 aatttgaacg ccagcacatg gactcgtcta ctagcgcagc ttaattaacc taggctgctg
     1081 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1141 ttttgctgaa acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata
     1201 aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga
     1261 caagctgacg accgggtctc cgcaagtggc acttttcggg gaaatgtgcg cggaacccct
     1321 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     1381 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     1441 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     1501 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     1561 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     1621 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     1681 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     1741 agcgagacga aatacgcggt cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     1801 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     1861 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     1921 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     1981 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     2041 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     2101 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     2161 gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt attgaagcat
     2221 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2281 aataggcatg ctagcgcaga aacgtcctag aagatgccag gaggatactt agcagagaga
     2341 caataaggcc ggagcgaagc cgtttttcca taggctccgc ccccctgacg aacatcacga
     2401 aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2461 tccccctgat ggctccctct tgcgctctcc tgttcccgtc ctgcggcgtc cgtgttgtgg
     2521 tggaggcttt acccaaatca ccacgtcccg ttccgtgtag acagttcgct ccaagctggg
     2581 ctgtgtgcaa gaaccccccg ttcagcccga ctgctgcgcc ttatccggta actatcatct
     2641 tgagtccaac ccggaaagac acgacaaaac gccactggca gcagccattg gtaactgaga
     2701 attagtggat ttagatatcg agagtcttga agtggtggcc taacagaggc tacactgaaa
     2761 ggacagtatt tggtatctgc gctccactaa agccagttac caggttaagc agttccccaa
     2821 ctgacttaac cttcgatcaa accgcctccc caggcggttt tttcgtttac agagcaggag
     2881 attacgacga tcgtaaaagg atctcaagaa gatcctttac ggattcccga caccatcact
     2941 ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata
     3001 taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     3061 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     3121 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3181 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     3241 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     3301 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     3361 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     3421 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     3481 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     3541 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     3601 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     3661 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     3721 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     3781 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     3841 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     3901 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     3961 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     4021 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     4081 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     4141 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     4201 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     4261 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     4321 actcctgcat taggaaatta atacgactca ctata
//
