LOCUS       sequence-417152-        6883 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of FIP200 NTD mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6883)
  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 6883)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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)-V44D-TSF
FEATURES             Location/Qualifiers
     source          1..6883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..641
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     intron          642..1658
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1582..1604
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1666..1685
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3665..3685
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3698..3721
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3758..3781
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3788..3811
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3904..3923)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3969..4024
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4023..4042)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4385..4401)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4385..4401)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4398..4420)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4409..4425
                     /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(4433..4439,4440..4457,4458..4463))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4478..4499
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4558..4753
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4604..4739
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4759..4893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4796..4815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4850..4869
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4961..4978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5132..5720)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5212..5231)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5891..6682,6683..6751))
                     /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     6514..6533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6752..6856)
                     /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 gggggggggg cggggggggg ggggggggcg aggcggagag
      541 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
      601 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      661 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      721 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      781 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      841 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      901 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      961 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
     1021 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
     1081 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
     1141 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
     1201 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
     1261 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
     1321 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
     1381 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
     1441 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1501 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1561 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     1621 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     1681 ttgtgctgtc tcatcatttt ggcaaagaat tcctcgatcg acggtatcga tagccaccat
     1741 gaagctctat gtgtttctcg tgaataccgg cactaccctg acctttgata ccgaactgac
     1801 cgtccagacc gtggctgacc tgaagcatgc tatccagtct aaatacaaga tcgcaattca
     1861 gcaccaggac ctggtggtca acggcggaga gtgcatggcc gctgatcgca gggtctgtac
     1921 ctattcagcc ggcaccgaca ctaacccaat cttcctcttt aataaggaga tgattctgtg
     1981 cgatagaccc cctgctatcc ccaagaccac ttttagcact gagaacgaca tggaaatcaa
     2041 agtggaggaa tccctgatga tgccagccgt gttccacacc gtcgcttctc gcactcagct
     2101 ggcactcgag atgtacgaag tggccaagaa actgtgctct ttctgtgagg gtctcgtcca
     2161 cgatgaacat ctgcagcacc agggctgggc agccattatg gctaacctgg aggactgttc
     2221 aaatagttat cagaaactgc tcttcaagtt tgagtctatc tactcaaact atctgcagag
     2281 catcgaagac attaaactga agctcacaca cctgggaacc gcagtgagcg tcatggccaa
     2341 gatccctctg ctcgagtgcc tgactaggca ttcctacaga gaatgtctgg gtaggctcga
     2401 tagtctgcca gaacacgagg acagcgagaa agccgaaatg aagcgctcta cagagctggt
     2461 gctctcacct gacatgccaa ggacaaccaa cgaaagtctg ctcaccagct tcccaaagtc
     2521 cgtggagcat gtctcccccg acactgccga tgctgaatct ggaaaggaga ttagagaaag
     2581 ttgccagagc accgtgcacc agcaggacga gactacaatt gacactaagg acggcgatct
     2641 gcccttcttt aacgtcagtc tgctcgattg gatcaacgtc caggacagac ctaatgatgt
     2701 ggagtccctg gtccggaagt gctttgatag catgtcccgt ctggacccac gtatcattcg
     2761 tcctttcatc gcagagtgtc gtcagaccat tgccaagctg gacaaccaga atatgaaagc
     2821 tattaagggt ctggaagatc ggctctacgc cctggaccag atgatcgcta gctgtggacg
     2881 cctggtgaac gagcagaagg aactcgctca gggtttcctg gcaaaccaga aaagagccga
     2941 gaatctcaag gatgcatcag tgctgcctga cctgtgcctc agtcatgcca atcagctgat
     3001 gattatgctc cagaaccacc gcaagctgct cgacatcaaa cagaagtgta ccactgctaa
     3061 acaggagctg gcaaacaatc tccatgtgag gctgaagtgg tgctgtttcg tcatgctgca
     3121 cgcagaccag gatggcgaga agctccaggc cctgctcagg ctggtgattg agctgctcga
     3181 aagagtgaag atcgtcgagg ctctgtccac cgtcccacag atgtactgcc tggcagtggt
     3241 cgaggtggtc agacgtaaaa tgtttatcaa gcactacaga gaatgggcag gtgccctcgt
     3301 gaaagatggc aagcgtctgt atgaagccga gaaatctaag cgtgagtcat tcggcaaact
     3361 gtttcggaag agcttcctcc gtaaccggct gttccgcgga ctcgacagtt ggccacccag
     3421 cttttgcacc cagaaaccta ggaagttcga ctgtgagctg ccagatatct ccctcaagga
     3481 cctgcagttt ctccagtcct tctgcccatc tgaggtgcag ccctttctga gggtccctct
     3541 gctctgtgac ttcgaacctc tgcaccagca tgtgctggcc ctccacaacc tcgtcaaggc
     3601 tgcacagagt ctggatgaga tgagccagac aatcaccgac ctgctcagcg aacagaaggg
     3661 taccgaaaac ctgtacttcc aatccggtat ggcggcctgg tcacacccgc agttcgagaa
     3721 aggtggaggt gcccgaggtg gatcgggagg tggatcgtgg agccacccgc agttcgaaaa
     3781 aggattcgat tacaaggatg acgatgacaa gtagctcgag gaattcactc ctcaggtgca
     3841 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     3901 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     3961 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     4021 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     4081 ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag
     4141 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     4201 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
     4261 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     4321 catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc ttggcgtaat
     4381 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4441 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4501 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca
     4561 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4621 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4681 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4741 aggcttttgc aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4801 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4861 aaactcatca atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg
     4921 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4981 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5041 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5101 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5161 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5221 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5281 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5341 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5401 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5461 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5521 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5581 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5641 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5701 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5761 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5821 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5881 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5941 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6001 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6061 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6121 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6181 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6241 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6301 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6361 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6421 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6481 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6541 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6601 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6661 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6721 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6781 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6841 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgg
//
