LOCUS       sequence-417176-        6903 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 6903)
  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 6903)
  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)-L324D/R543D/R548D-TSF
FEATURES             Location/Qualifiers
     source          1..6903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3685..3705
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3718..3741
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3778..3801
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3808..3831
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3924..3943)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3989..4044
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4043..4062)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4405..4421)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4405..4421)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4418..4440)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4429..4445
                     /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(4453..4459,4460..4477,4478..4483))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4498..4519
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4578..4773
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4624..4759
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4779..4913
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4816..4835)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4870..4889
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4981..4998)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5152..5740)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5232..5251)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5911..6702,6703..6771))
                     /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     6534..6553
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6772..6876)
                     /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 gggggcgcgc gccaggsggg gggggggggg gsgagggggg
      541 gggsggggsg aggsggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg rgggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcctcgatcg
     1741 acggtatcga tagccaccat gaagctctat gtgtttctcg tgaataccgg cactaccctg
     1801 acctttgata ccgaactgac cgtccagacc gtggctgacc tgaagcatgc tatccagtct
     1861 aaatacaaga tcgcaattca gcaccaggtg ctggtggtca acggcggaga gtgcatggcc
     1921 gctgatcgca gggtctgtac ctattcagcc ggcaccgaca ctaacccaat cttcctcttt
     1981 aataaggaga tgattctgtg cgatagaccc cctgctatcc ccaagaccac ttttagcact
     2041 gagaacgaca tggaaatcaa agtggaggaa tccctgatga tgccagccgt gttccacacc
     2101 gtcgcttctc gcactcagct ggcactcgag atgtacgaag tggccaagaa actgtgctct
     2161 ttctgtgagg gtctcgtcca cgatgaacat ctgcagcacc agggctgggc agccattatg
     2221 gctaacctgg aggactgttc aaatagttat cagaaactgc tcttcaagtt tgagtctatc
     2281 tactcaaact atctgcagag catcgaagac attaaactga agctcacaca cctgggaacc
     2341 gcagtgagcg tcatggccaa gatccctctg ctcgagtgcc tgactaggca ttcctacaga
     2401 gaatgtctgg gtaggctcga tagtctgcca gaacacgagg acagcgagaa agccgaaatg
     2461 aagcgctcta cagagctggt gctctcacct gacatgccaa ggacaaccaa cgaaagtctg
     2521 ctcaccagct tcccaaagtc cgtggagcat gtctcccccg acactgccga tgctgaatct
     2581 ggaaaggaga ttagagaaag ttgccagagc accgtgcacc agcaggacga gactacaatt
     2641 gacactaagg acggcgatct gcccttcttt aacgtcagtc tgctcgattg gatcaacgtc
     2701 caggacagac ctaatgatgt ggagtccgac gtccggaagt gctttgatag catgtcccgt
     2761 ctggacccac gtatcattcg tcctttcatc gcagagtgtc gtcagaccat tgccaagctg
     2821 gacaaccaga atatgaaagc tattaagggt ctggaagatc ggctctacgc cctggaccag
     2881 atgatcgcta gctgtggacg cctggtgaac gagcagaagg aactcgctca gggtttcctg
     2941 gcaaaccaga aaagagccga gaatctcaag gatgcatcag tgctgcctga cctgtgcctc
     3001 agtcatgcca atcagctgat gattatgctc cagaaccacc gcaagctgct cgacatcaaa
     3061 cagaagtgta ccactgctaa acaggagctg gcaaacaatc tccatgtgag gctgaagtgg
     3121 tgctgtttcg tcatgctgca cgcagaccag gatggcgaga agctccaggc cctgctcagg
     3181 ctggtgattg agctgctcga aagagtgaag atcgtcgagg ctctgtccac cgtcccacag
     3241 atgtactgcc tggcagtggt cgaggtggtc agacgtaaaa tgtttatcaa gcactacaga
     3301 gaatgggcag gtgccctcgt gaaagatggc aagcgtctgt atgaagccga gaaatctaag
     3361 cgtgagtcat tcggcaaact gtttgacaag agcttcctcg ataaccggct gttccgcgga
     3421 ctcgacagtt ggccacccag cttttgcacc cagaaaccta ggaagttcga ctgtgagctg
     3481 ccagatatct ccctcaagga cctgcagttt ctccagtcct tctgcccatc tgaggtgcag
     3541 ccctttctga gggtccctct gctctgtgac ttcgaacctc tgcaccagca tgtgctggcc
     3601 ctccacaacc tcgtcaaggc tgcacagagt ctggatgaga tgagccagac aatcaccgac
     3661 ctgctcagcg aacagaaggg taccgaaaac ctgtacttcc aatccggtat ggcggcctgg
     3721 tcacacccgc agttcgagaa aggtggaggt gcccgaggtg gatcgggagg tggatcgtgg
     3781 agccacccgc agttcgaaaa aggattcgat tacaaggatg acgatgacaa gtagctcgag
     3841 gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     3901 ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg
     3961 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     4021 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     4081 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     4141 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     4201 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     4261 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     4321 ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg
     4381 cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4441 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4501 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4561 gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4621 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4681 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4741 ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata
     4801 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4861 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atccgctgca
     4921 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4981 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5041 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5101 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5161 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5221 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5281 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5341 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5401 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5461 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5521 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5581 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5641 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5701 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5761 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5821 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5881 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5941 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6001 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6061 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6121 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6181 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6241 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6301 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6361 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6421 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6481 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6541 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6601 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6661 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6721 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6781 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6841 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6901 tgg
//
