LOCUS       sequence-400892-        7918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FIP200 NTD-GFP mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7918)
  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 7918)
  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)-MBP
FEATURES             Location/Qualifiers
     source          1..7918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..657
                     /label=chicken beta-actin promoter
     intron          658..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3680..3700
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3704..4804
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     4778..4801
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4814..4843
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(4936..4955)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5001..5056
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5055..5074)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5417..5433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5417..5433)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5430..5452)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5441..5457
                     /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(5465..5471,5472..5489,5490..5495))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5510..5531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5590..5785
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5636..5771
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5791..5925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5828..5847)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5882..5901
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5993..6010)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6164..6752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6244..6263)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6923..7714,7715..7783))
                     /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     7546..7565
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7784..7888)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gggcgcgcca ggcggggcgg ggcggggcga ggggcggggc
      541 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      601 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      661 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      721 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      781 ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc
      841 cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg
      901 tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc
      961 gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
     1081 gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcct cgatcgacgg
     1741 tatcgatgcc accatgaagc tctatgtgtt tctcgtgaat accggcacta ccctgacctt
     1801 tgataccgaa ctgaccgtcc agaccgtggc tgacctgaag catgctatcc agtctaaata
     1861 caagatcgca attcagcacc aggtgctggt ggtcaacggc ggagagtgca tggccgctga
     1921 tcgcagggtc tgtacctatt cagccggcac cgacactaac ccaatcttcc tctttaataa
     1981 ggagatgatt ctgtgcgata gaccccctgc tatccccaag accactttta gcactgagaa
     2041 cgacatggaa atcaaagtgg aggaatccct gatgatgcca gccgtgttcc acaccgtcgc
     2101 ttctcgcact cagctggcac tcgagatgta cgaagtggcc aagaaactgt gctctttctg
     2161 tgagggtctc gtccacgatg aacatctgca gcaccagggc tgggcagcca ttatggctaa
     2221 cctggaggac tgttcaaata gttatcagaa actgctcttc aagtttgagt ctatctactc
     2281 aaactatctg cagagcatcg aagacattaa actgaagctc acacacctgg gaaccgcagt
     2341 gagcgtcatg gccaagatcc ctctgctcga gtgcctgact aggcattcct acagagaatg
     2401 tctgggtagg ctcgatagtc tgccagaaca cgaggacagc gagaaagccg aaatgaagcg
     2461 ctctacagag ctggtgctct cacctgacat gccaaggaca accaacgaaa gtctgctcac
     2521 cagcttccca aagtccgtgg agcatgtctc ccccgacact gccgatgctg aatctggaaa
     2581 ggagattaga gaaagttgcc agagcaccgt gcaccagcag gacgagacta caattgacac
     2641 taaggacggc gatctgccct tctttaacgt cagtctgctc gattggatca acgtccagga
     2701 cagacctaat gatgtggagt ccctggtccg gaagtgcttt gatagcatgt cccgtctgga
     2761 cccacgtatc attcgtcctt tcatcgcaga gtgtcgtcag accattgcca agctggacaa
     2821 ccagaatatg aaagctatta agggtctgga agatcggctc tacgccctgg accagatgat
     2881 cgctagctgt ggacgcctgg tgaacgagca gaaggaactc gctcagggtt tcctggcaaa
     2941 ccagaaaaga gccgagaatc tcaaggatgc atcagtgctg cctgacctgt gcctcagtca
     3001 tgccaatcag ctgatgatta tgctccagaa ccaccgcaag ctgctcgaca tcaaacagaa
     3061 gtgtaccact gctaaacagg agctggcaaa caatctccat gtgaggctga agtggtgctg
     3121 tttcgtcatg ctgcacgcag accaggatgg cgagaagctc caggccctgc tcaggctggt
     3181 gattgagctg ctcgaaagag tgaagatcgt cgaggctctg tccaccgtcc cacagatgta
     3241 ctgcctggca gtggtcgagg tggtcagacg taaaatgttt atcaagcact acagagaatg
     3301 ggcaggtgcc ctcgtgaaag atggcaagcg tctgtatgaa gccgagaaat ctaagcgtga
     3361 gtcattcggc aaactgtttc ggaagagctt cctccgtaac cggctgttcc gcggactcga
     3421 cagttggcca cccagctttt gcacccagaa acctaggaag ttcgactgtg agctgccaga
     3481 tatctccctc aaggacctgc agtttctcca gtccttctgc ccatctgagg tgcagccctt
     3541 tctgagggtc cctctgctct gtgacttcga acctctgcac cagcatgtgc tggccctcca
     3601 caacctcgtc aaggctgcac agagtctgga tgagatgagc cagacaatca ccgacctgct
     3661 cagcgaacag aagggtaccg aaaacctgta cttccaatcc ggtatgaaaa tcgaagaagg
     3721 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
     3781 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
     3841 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
     3901 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
     3961 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
     4021 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
     4081 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
     4141 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
     4201 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
     4261 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
     4321 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
     4381 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     4441 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     4501 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     4561 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     4621 gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca
     4681 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     4741 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     4801 gactggcagt agccaccatc accatcacca tcatcaccat cactagctcg aggaattcac
     4861 tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca
     4921 caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct
     4981 tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa
     5041 ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa
     5101 tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg
     5161 ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca
     5221 tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc
     5281 tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg
     5341 actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa
     5401 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5461 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5521 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5581 agcggatccg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     5641 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     5701 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     5761 ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac
     5821 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5881 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa
     5941 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6001 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6061 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6121 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6181 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6241 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6301 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6361 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6421 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6481 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6541 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6601 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6661 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6721 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6781 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6841 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6901 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6961 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7021 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7081 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7141 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7201 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7261 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7321 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7381 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7441 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7501 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7561 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7621 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7681 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7741 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7801 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7861 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgggtc
//
