LOCUS       sequence-401048-        6901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FIP200 NTD-TwinStrep-Flag mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6901)
  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 6901)
  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)-TSF
FEATURES             Location/Qualifiers
     source          1..6901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..456
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        458..733
                     /label=chicken beta-actin promoter
     intron          734..1750
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1674..1696
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1758..1777
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3756..3776
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3789..3812
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3849..3872
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3879..3902
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3995..4014)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4060..4115
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4114..4133)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4476..4492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4476..4492)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4489..4511)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4500..4516
                     /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(4524..4530,4531..4548,4549..4554))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4569..4590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4649..4844
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4695..4830
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4850..4984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4887..4906)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4941..4960
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5052..5069)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5223..5811)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5303..5322)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5982..6773,6774..6842))
                     /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     6605..6624
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6843..6901,1..46))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
       61 aagtgccacc tgggtcgaca ttgattattg actagttatt aatagtaatc aattacgggg
      121 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      181 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      241 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      301 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      361 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      421 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      481 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      541 ttattttgtg cagcgatggg ggcggggggg gggggggsgc gcgccaggcg gggcggggcg
      601 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
      661 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
      721 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
      781 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
      841 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
      901 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc
      961 ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg
     1021 cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg
     1081 gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg
     1141 cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca
     1201 accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct
     1261 ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt
     1321 gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc
     1381 ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc
     1441 gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag
     1501 gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga
     1561 aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc
     1621 tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg
     1681 cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt
     1741 tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga
     1801 attcctcgat cgacggtatc gatgccacca tgaagctcta tgtgtttctc gtgaataccg
     1861 gcactaccct gacctttgat accgaactga ccgtccagac cgtggctgac ctgaagcatg
     1921 ctatccagtc taaatacaag atcgcaattc agcaccaggt gctggtggtc aacggcggag
     1981 agtgcatggc cgctgatcgc agggtctgta cctattcagc cggcaccgac actaacccaa
     2041 tcttcctctt taataaggag atgattctgt gcgatagacc ccctgctatc cccaagacca
     2101 cttttagcac tgagaacgac atggaaatca aagtggagga atccctgatg atgccagccg
     2161 tgttccacac cgtcgcttct cgcactcagc tggcactcga gatgtacgaa gtggccaaga
     2221 aactgtgctc tttctgtgag ggtctcgtcc acgatgaaca tctgcagcac cagggctggg
     2281 cagccattat ggctaacctg gaggactgtt caaatagtta tcagaaactg ctcttcaagt
     2341 ttgagtctat ctactcaaac tatctgcaga gcatcgaaga cattaaactg aagctcacac
     2401 acctgggaac cgcagtgagc gtcatggcca agatccctct gctcgagtgc ctgactaggc
     2461 attcctacag agaatgtctg ggtaggctcg atagtctgcc agaacacgag gacagcgaga
     2521 aagccgaaat gaagcgctct acagagctgg tgctctcacc tgacatgcca aggacaacca
     2581 acgaaagtct gctcaccagc ttcccaaagt ccgtggagca tgtctccccc gacactgccg
     2641 atgctgaatc tggaaaggag attagagaaa gttgccagag caccgtgcac cagcaggacg
     2701 agactacaat tgacactaag gacggcgatc tgcccttctt taacgtcagt ctgctcgatt
     2761 ggatcaacgt ccaggacaga cctaatgatg tggagtccct ggtccggaag tgctttgata
     2821 gcatgtcccg tctggaccca cgtatcattc gtcctttcat cgcagagtgt cgtcagacca
     2881 ttgccaagct ggacaaccag aatatgaaag ctattaaggg tctggaagat cggctctacg
     2941 ccctggacca gatgatcgct agctgtggac gcctggtgaa cgagcagaag gaactcgctc
     3001 agggtttcct ggcaaaccag aaaagagccg agaatctcaa ggatgcatca gtgctgcctg
     3061 acctgtgcct cagtcatgcc aatcagctga tgattatgct ccagaaccac cgcaagctgc
     3121 tcgacatcaa acagaagtgt accactgcta aacaggagct ggcaaacaat ctccatgtga
     3181 ggctgaagtg gtgctgtttc gtcatgctgc acgcagacca ggatggcgag aagctccagg
     3241 ccctgctcag gctggtgatt gagctgctcg aaagagtgaa gatcgtcgag gctctgtcca
     3301 ccgtcccaca gatgtactgc ctggcagtgg tcgaggtggt cagacgtaaa atgtttatca
     3361 agcactacag agaatgggca ggtgccctcg tgaaagatgg caagcgtctg tatgaagccg
     3421 agaaatctaa gcgtgagtca ttcggcaaac tgtttcggaa gagcttcctc cgtaaccggc
     3481 tgttccgcgg actcgacagt tggccaccca gcttttgcac ccagaaacct aggaagttcg
     3541 actgtgagct gccagatatc tccctcaagg acctgcagtt tctccagtcc ttctgcccat
     3601 ctgaggtgca gccctttctg agggtccctc tgctctgtga cttcgaacct ctgcaccagc
     3661 atgtgctggc cctccacaac ctcgtcaagg ctgcacagag tctggatgag atgagccaga
     3721 caatcaccga cctgctcagc gaacagaagg gtaccgaaaa cctgtacttc caatccggta
     3781 tggcggcctg gtcacacccg cagttcgaga aaggtggagg tgcccgaggt ggatcgggag
     3841 gtggatcgtg gagccacccg cagttcgaaa aaggattcga ttacaaggat gacgatgaca
     3901 agtagctcga ggaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
     3961 tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg
     4021 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     4081 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     4141 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc
     4201 tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg
     4261 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt
     4321 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca
     4381 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat
     4441 ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4501 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4561 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4621 tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc
     4681 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     4741 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4801 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat
     4861 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4921 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4981 gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5041 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5101 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5161 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5221 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5281 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5341 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5401 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5461 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5521 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5581 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5641 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5701 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5761 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5821 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5881 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5941 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6001 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6061 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6121 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6181 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6241 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6301 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6361 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6421 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6481 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6541 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6601 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6661 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6721 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6781 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6841 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6901 t
//
