LOCUS       sequence-267687-        6160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  stably express FBR-Fis1 in mammalian cells with puromycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6160)
  AUTHORS   Vargas JNS, Wang C, Bunker E, Hao L, Maric D, Schiavo G, Randow F, 
            Youle RJ
  TITLE     Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during 
            Selective Autophagy.
  JOURNAL   Mol Cell. 2019 Apr 18;74(2):347-362.e6. doi: 
            10.1016/j.molcel.2019.02.010. Epub 2019 Mar 7.
  PUBMED    30853401
REFERENCE   2  (bases 1 to 6160)
  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: 
            "10.1016/j.molcel.2019.02.010"; journalName: "Mol Cell"; date: 
            "2019-04-18- 18"; volume: "74"; issue: "2"; pages: "347-362.e6"
COMMENT     Sequence Label: pBMN-puro-P2A-FRB-Fis1
FEATURES             Location/Qualifiers
     source          1..6160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..470
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    535..892
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             957..1373
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1303..1325
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1341..1357
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1383..1757
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1695..1711
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             1800..2396
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1800..1819)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2296..2316
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2406..2462
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2481..2759
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     LTR             3137..3606
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3736..3753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3907..4495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3987..4006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4659..5450,5451..5519))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5282..5301
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5520..5624)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
       61 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
      121 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
      181 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
      241 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
      301 aaaagagccc acaacccctc actcggcgcg ccagtcctcc gattgactga gtcgcccggg
      361 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
      421 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
      481 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg
      541 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgactgatt ttatgcgcct
      601 gcgtcggtac tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga
      661 gttcggaaca cccggccgca accctgggag acgtcccagg gacttcgggg gccgtttttg
      721 tggcccgacc tgagtccaaa aatcccgatc gttttggact ctttggtgca ccccccttag
      781 aggagggata tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg
      841 aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg tcttgtctgc tgcagcatcg
      901 ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc tgaaaatatg ggcccgggcc
      961 agactgttac cactccctta agtttgacct taggtcactg gaaagatgtc gagcggatcg
     1021 ctcacaacca gtcggtagat gtcaagaaga gacgttgggt taccttctgc tctgcagaat
     1081 ggccaacctt taacgtcgga tggccgcgag acggcacctt taaccgagac ctcatcaccc
     1141 aggttaagat caaggtcttt tcacctggcc cgcatggaca cccagaccag gtcccctaca
     1201 tcgtgacctg ggaagccttg gcttttgacc cccctccctg ggtcaagccc tttgtacacc
     1261 ctaagcctcc gcctcctctt cctccatccg ccccgtctct cccccttgaa cctcctcgtt
     1321 cgaccccgcc tcgatcctcc ctttatccag ccctcactcc ttctctaggc gcccccatat
     1381 ggccatatga gatcttatat ggggcacccc cgccccttgt aaacttccct gaccctgaca
     1441 tgacaagagt tactaacagc ccctctctcc aagctcactt acaggctctc tacttagtcc
     1501 agcacgaagt ctggagacct ctggcggcag cctaccaaga acaactggac cgaccggtgg
     1561 tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg ccgacaccag actaagaacc
     1621 tagaacctcg ctggaaagga ccttacacag tcctgctgac cacccccacc gccctcaaag
     1681 tagacggcat cgcagcttgg atacacgccg cccacgtgaa ggctgccgac cccgggggtg
     1741 gaccatcctc tagactgccg gatcccagtg tggtggtacg ggaattcaag cttgccacca
     1801 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     1861 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     1921 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     1981 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     2041 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     2101 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     2161 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca
     2221 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     2281 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     2341 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgccggaa
     2401 gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     2461 ctctcgagat ggcttctaga atcctctggc atgagatgtg gcatgaaggc ctggaagagg
     2521 catctcgttt gtactttggg gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct
     2581 tgcatgctat gatggaacgg ggcccccaga ctctgaagga aacatccttt aatcaggcct
     2641 atggtcgaga tttaatggag gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg
     2701 tcaaggacct cctccaagcc tgggacctct attatcatgt gttccgacga atctcaaaga
     2761 ctagttacgt ccgcgggttg ctgcagacag agccccagaa caaccaggcc aaggaactgg
     2821 agcggctcat tgacaaggcc atgaagaaag atggactcgt gggcatggcc atcgtgggag
     2881 gcatggccct gggtgtggcg ggactggccg gactcatcgg acttgctgtg tccaagtcca
     2941 aatcctgagc ggccgccagc acagtggtcg acgataaaat aaaagatttt atttagtctc
     3001 cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg
     3061 ccattttgca aggcatggaa aaatacataa ctgagaatag agaagttcag atcaaggtca
     3121 ggaacagatg gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     3181 cccggctcag ggccaagaac agatggaaca gctgaatatg ggccaaacag gatatctgtg
     3241 gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtccagc
     3301 cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga
     3361 ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct
     3421 gctccccgag ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt
     3481 gactgagtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg
     3541 tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt
     3601 ctttcacatg cagcatgtat caaaattaat ttggtttttt ttcttaagta tttacattaa
     3661 atggccatag ttgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3721 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3781 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3841 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3901 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3961 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4021 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4081 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4141 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4201 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4261 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4321 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4381 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4441 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4501 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4561 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttgcggc cggccgcaaa
     4621 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4681 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4741 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4801 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4861 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4921 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4981 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5041 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5101 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5161 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5221 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5281 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5341 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5401 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5461 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5521 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5581 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5641 gtgccaccag ctttgctctt aggagtttcc taatacatcc caaactcaaa tatataaagc
     5701 atttgacttg ttctatgccc tagggggcgg ggggaagcta agccagcttt ttttaacatt
     5761 taaaatgtta attccatttt aaatgcacag atgtttttat ttcataaggg tttcaatgtg
     5821 catgaatgct gcaatattcc tgttaccaaa gctagtataa ataaaaatag ataaacgtgg
     5881 aaattactta gagtttctgt cattaacgtt tccttcctca gttgacaaca taaatgcgct
     5941 gctgagaagc cagtttgcat ctgtcaggat caatttccca ttatgccagt catattaatt
     6001 actagtcaat tagttgattt ttatttttga catatacatg tgaaagaccc cacctgtagg
     6061 tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga
     6121 atagaaaagt tcagatcaag gtcaggaaca gatggaacag
//
