LOCUS       sequence-400890-        7529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FIP200 NTD-GFP-10His mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7529)
  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 7529)
  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)-msfGFP
FEATURES             Location/Qualifiers
     source          1..7529
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        127..506
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        508..783
                     /label=chicken beta-actin promoter
     intron          784..1800
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1724..1746
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1808..1827
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3807..3827
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(3834..3836,3837..3839,3840..4550)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3879..3900)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4487..4508
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4551..4580
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(4673..4692)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4738..4793
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4792..4811)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5154..5170)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5154..5170)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5167..5189)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5178..5194
                     /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(5202..5208,5209..5226,5227..5232))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5247..5268
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5327..5522
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5373..5508
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5528..5662
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5565..5584)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5619..5638
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5730..5747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5901..6489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5981..6000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6660..7451,7452..7520))
                     /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     7283..7302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7521..7529,1..96))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
       61 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
      121 tgggtcgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      181 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      241 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      301 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      361 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      421 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      481 acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc
      541 ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg
      601 cagcgatggg ggcggggggg gggggggsgc gcgccaggcg gggcggggcg gggcgagggg
      661 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
      721 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
      781 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      841 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      901 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      961 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
     1021 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
     1081 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1141 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     1201 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1261 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1321 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     1381 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
     1441 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1501 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1561 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1621 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1681 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1741 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1801 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgat
     1861 cgacggtatc gatagccacc atgaagctct atgtgtttct cgtgaatacc ggcactaccc
     1921 tgacctttga taccgaactg accgtccaga ccgtggctga cctgaagcat gctatccagt
     1981 ctaaatacaa gatcgcaatt cagcaccagg tgctggtggt caacggcgga gagtgcatgg
     2041 ccgctgatcg cagggtctgt acctattcag ccggcaccga cactaaccca atcttcctct
     2101 ttaataagga gatgattctg tgcgatagac cccctgctat ccccaagacc acttttagca
     2161 ctgagaacga catggaaatc aaagtggagg aatccctgat gatgccagcc gtgttccaca
     2221 ccgtcgcttc tcgcactcag ctggcactcg agatgtacga agtggccaag aaactgtgct
     2281 ctttctgtga gggtctcgtc cacgatgaac atctgcagca ccagggctgg gcagccatta
     2341 tggctaacct ggaggactgt tcaaatagtt atcagaaact gctcttcaag tttgagtcta
     2401 tctactcaaa ctatctgcag agcatcgaag acattaaact gaagctcaca cacctgggaa
     2461 ccgcagtgag cgtcatggcc aagatccctc tgctcgagtg cctgactagg cattcctaca
     2521 gagaatgtct gggtaggctc gatagtctgc cagaacacga ggacagcgag aaagccgaaa
     2581 tgaagcgctc tacagagctg gtgctctcac ctgacatgcc aaggacaacc aacgaaagtc
     2641 tgctcaccag cttcccaaag tccgtggagc atgtctcccc cgacactgcc gatgctgaat
     2701 ctggaaagga gattagagaa agttgccaga gcaccgtgca ccagcaggac gagactacaa
     2761 ttgacactaa ggacggcgat ctgcccttct ttaacgtcag tctgctcgat tggatcaacg
     2821 tccaggacag acctaatgat gtggagtccc tggtccggaa gtgctttgat agcatgtccc
     2881 gtctggaccc acgtatcatt cgtcctttca tcgcagagtg tcgtcagacc attgccaagc
     2941 tggacaacca gaatatgaaa gctattaagg gtctggaaga tcggctctac gccctggacc
     3001 agatgatcgc tagctgtgga cgcctggtga acgagcagaa ggaactcgct cagggtttcc
     3061 tggcaaacca gaaaagagcc gagaatctca aggatgcatc agtgctgcct gacctgtgcc
     3121 tcagtcatgc caatcagctg atgattatgc tccagaacca ccgcaagctg ctcgacatca
     3181 aacagaagtg taccactgct aaacaggagc tggcaaacaa tctccatgtg aggctgaagt
     3241 ggtgctgttt cgtcatgctg cacgcagacc aggatggcga gaagctccag gccctgctca
     3301 ggctggtgat tgagctgctc gaaagagtga agatcgtcga ggctctgtcc accgtcccac
     3361 agatgtactg cctggcagtg gtcgaggtgg tcagacgtaa aatgtttatc aagcactaca
     3421 gagaatgggc aggtgccctc gtgaaagatg gcaagcgtct gtatgaagcc gagaaatcta
     3481 agcgtgagtc attcggcaaa ctgtttcgga agagcttcct ccgtaaccgg ctgttccgcg
     3541 gactcgacag ttggccaccc agcttttgca cccagaaacc taggaagttc gactgtgagc
     3601 tgccagatat ctccctcaag gacctgcagt ttctccagtc cttctgccca tctgaggtgc
     3661 agccctttct gagggtccct ctgctctgtg acttcgaacc tctgcaccag catgtgctgg
     3721 ccctccacaa cctcgtcaag gctgcacaga gtctggatga gatgagccag acaatcaccg
     3781 acctgctcag cgaacagaag ggtaccgaaa acctgtactt ccaatccggt tctatggtga
     3841 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     3901 taaacggcca caagttcagc gtgcgcggcg agggcgaggg cgatgccacc aacggcaagc
     3961 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     4021 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     4081 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc tccttcaagg
     4141 acgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     4201 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     4261 agtacaactt caacagccac aacgtctata tcacggccga caagcagaag aacggcatca
     4321 aggcgaactt caagatccgc cacaacgtcg aggacggcag cgtgcagctc gccgaccact
     4381 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     4441 gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     4501 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag catcaccatc
     4561 accatcacca tcaccatcac tagctcgagg aattcactcc tcaggtgcag gctgcctatc
     4621 agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc
     4681 cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact tctggctaat
     4741 aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa
     4801 ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg
     4861 caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt
     4921 atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt
     4981 agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct
     5041 tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc
     5101 ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc atggtcatag
     5161 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5221 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5281 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat ctcaattagt
     5341 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     5401 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     5461 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5521 aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5581 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5641 tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc
     5701 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5761 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5821 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5881 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5941 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6001 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6061 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6121 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6181 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6241 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6301 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6361 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6421 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6481 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6541 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6601 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6661 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6721 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6781 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6841 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6901 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6961 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7021 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7081 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7141 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7201 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7261 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7321 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7381 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7441 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7501 cggaaatgtt gaatactcat actcttcct
//
