LOCUS       sequence-417175-        6905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of FIP200 NTD mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6905)
  AUTHORS   Chen M, Ren X, Nguyen TN, Khuu G, Cook ASI, Lazarou M, Hurley JH
  TITLE     Structure and activation of the human autophagy-initiating 
            ULK1C:PI3KC3-C1 supercomplex
  JOURNAL   bioRxiv 2023.06.01.543278
REFERENCE   2  (bases 1 to 6905)
  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"; journalName: "bioRxiv 
            2023.06.01.543278"
COMMENT     Sequence Label: pCAG-FIP200 (1-640)-R334D-TSF
FEATURES             Location/Qualifiers
     source          1..6905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..663
                     /label=chicken beta-actin promoter
     intron          664..1680
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1604..1626
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1688..1707
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3687..3707
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3720..3743
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3780..3803
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3810..3833
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3926..3945)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3991..4046
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4045..4064)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4407..4423)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4407..4423)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4420..4442)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4431..4447
                     /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        complement(join(4455..4461,4462..4479,4480..4485))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4500..4521
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4580..4775
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4626..4761
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4781..4915
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4818..4837)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4872..4891
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4983..5000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5154..5742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5234..5253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5913..6704,6705..6773))
                     /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     6536..6555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6774..6878)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggggcg cgccaggggg gggggggggg gggggggggg
      541 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
      601 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
      661 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      721 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      781 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      841 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
      961 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1021 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     1081 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1141 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1201 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     1261 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
     1321 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1381 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1441 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1501 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1561 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1621 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1681 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgat
     1741 cgacggtatc gatagccacc atgaagctct atgtgtttct cgtgaatacc ggcactaccc
     1801 tgacctttga taccgaactg accgtccaga ccgtggctga cctgaagcat gctatccagt
     1861 ctaaatacaa gatcgcaatt cagcaccagg tgctggtggt caacggcgga gagtgcatgg
     1921 ccgctgatcg cagggtctgt acctattcag ccggcaccga cactaaccca atcttcctct
     1981 ttaataagga gatgattctg tgcgatagac cccctgctat ccccaagacc acttttagca
     2041 ctgagaacga catggaaatc aaagtggagg aatccctgat gatgccagcc gtgttccaca
     2101 ccgtcgcttc tcgcactcag ctggcactcg agatgtacga agtggccaag aaactgtgct
     2161 ctttctgtga gggtctcgtc cacgatgaac atctgcagca ccagggctgg gcagccatta
     2221 tggctaacct ggaggactgt tcaaatagtt atcagaaact gctcttcaag tttgagtcta
     2281 tctactcaaa ctatctgcag agcatcgaag acattaaact gaagctcaca cacctgggaa
     2341 ccgcagtgag cgtcatggcc aagatccctc tgctcgagtg cctgactagg cattcctaca
     2401 gagaatgtct gggtaggctc gatagtctgc cagaacacga ggacagcgag aaagccgaaa
     2461 tgaagcgctc tacagagctg gtgctctcac ctgacatgcc aaggacaacc aacgaaagtc
     2521 tgctcaccag cttcccaaag tccgtggagc atgtctcccc cgacactgcc gatgctgaat
     2581 ctggaaagga gattagagaa agttgccaga gcaccgtgca ccagcaggac gagactacaa
     2641 ttgacactaa ggacggcgat ctgcccttct ttaacgtcag tctgctcgat tggatcaacg
     2701 tccaggacag acctaatgat gtggagtccc tggtccggaa gtgctttgat agcatgtccg
     2761 atctggaccc acgtatcatt cgtcctttca tcgcagagtg tcgtcagacc attgccaagc
     2821 tggacaacca gaatatgaaa gctattaagg gtctggaaga tcggctctac gccctggacc
     2881 agatgatcgc tagctgtgga cgcctggtga acgagcagaa ggaactcgct cagggtttcc
     2941 tggcaaacca gaaaagagcc gagaatctca aggatgcatc agtgctgcct gacctgtgcc
     3001 tcagtcatgc caatcagctg atgattatgc tccagaacca ccgcaagctg ctcgacatca
     3061 aacagaagtg taccactgct aaacaggagc tggcaaacaa tctccatgtg aggctgaagt
     3121 ggtgctgttt cgtcatgctg cacgcagacc aggatggcga gaagctccag gccctgctca
     3181 ggctggtgat tgagctgctc gaaagagtga agatcgtcga ggctctgtcc accgtcccac
     3241 agatgtactg cctggcagtg gtcgaggtgg tcagacgtaa aatgtttatc aagcactaca
     3301 gagaatgggc aggtgccctc gtgaaagatg gcaagcgtct gtatgaagcc gagaaatcta
     3361 agcgtgagtc attcggcaaa ctgtttcgga agagcttcct ccgtaaccgg ctgttccgcg
     3421 gactcgacag ttggccaccc agcttttgca cccagaaacc taggaagttc gactgtgagc
     3481 tgccagatat ctccctcaag gacctgcagt ttctccagtc cttctgccca tctgaggtgc
     3541 agccctttct gagggtccct ctgctctgtg acttcgaacc tctgcaccag catgtgctgg
     3601 ccctccacaa cctcgtcaag gctgcacaga gtctggatga gatgagccag acaatcaccg
     3661 acctgctcag cgaacagaag ggtaccgaaa acctgtactt ccaatccggt atggcggcct
     3721 ggtcacaccc gcagttcgag aaaggtggag gtgcccgagg tggatcggga ggtggatcgt
     3781 ggagccaccc gcagttcgaa aaaggattcg attacaagga tgacgatgac aagtagctcg
     3841 aggaattcac tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg
     3901 ccctggctca caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca
     3961 tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag
     4021 tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa
     4081 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca
     4141 tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt
     4201 ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt
     4261 tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc
     4321 tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc
     4381 tgcagcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4441 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4501 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4561 ctgtcgtgcc agcggatccg catctcaatt agtcagcaac catagtcccg cccctaactc
     4621 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4681 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4741 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta
     4801 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4861 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg
     4921 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4981 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5041 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5101 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5161 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5221 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5281 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5341 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5401 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5461 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5521 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5581 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5641 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5701 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5761 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5821 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5881 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5941 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6001 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6061 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6121 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6181 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6241 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6301 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6361 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6421 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6481 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6541 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6601 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6661 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6721 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6781 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6841 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6901 cctgg
//
