LOCUS       sequence-501940-        7660 bp    DNA     circular SYN 04-MAR-2026
DEFINITION  Transient mammalian expression of GFP-RavZ C258A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7660)
  AUTHORS   Fuller DM, Wu Y, Schueder F, Rasool B, Nag S, Korfhage JL, 
            Garcia-Milian R, Melnyk KD, Bewersdorf J, De Camilli P, Melia TJ
  TITLE     ATG2A engages RAB1A and ARFGAP1 positive membranes during 
            autophagosome biogenesis
  JOURNAL   eLife 2025 14:RP107316
REFERENCE   2  (bases 1 to 7660)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 4, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "eLife 2025 
            14:RP107316"
COMMENT     Sequence Label: GFP-RavZ C258A
FEATURES             Location/Qualifiers
     source          1..7660
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(34..62)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     657..677
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    739..763
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2291..2315)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2347..2364)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2353..2577
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2623..3051
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2710..2729)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2920..2941
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3060..3080)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3065..3394
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3245..3380
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3307..3326
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3461..4255
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3515..3534)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4125..4144
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4429..4550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4466..4485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4520..4539
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4599..4615)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4599..4615)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4612..4634)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4623..4639
                     /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(4647..4653,4654..4671,4672..4677))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4692..4713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4830..4847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5001..5589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5081..5100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5760..6551,6552..6620))
                     /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     6383..6402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6621..6725)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6800..6819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6991..7370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7371..7574
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7525..7545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7619..7638
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7619..7637
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 atggccagca aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat
       61 ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgctacatac
      121 ggaaagctta cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca
      181 cttgtcacta ctttctctta tggtgttcaa tgcttttccc gttatccgga tcatatgaaa
      241 cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaacg cactatatct
      301 ttcaaagatg acgggaacta caagacgcgt gctgaagtca agtttgaagg tgataccctt
      361 gttaatcgta tcgagttaaa aggtattgat tttaaagaag atggaaacat tctcggacac
      421 aaactcgagt acaactataa ctcacacaat gtatacatca cggcagacaa acaaaagaat
      481 ggaatcaaag ctaacttcaa aattcgccac aacattgaag atggatccgt tcaactagca
      541 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat
      601 tacctgtcga cacaatctgc cctttcgaaa gatcccaacg aaaagcgtga ccacatggtc
      661 cttcttgagt ttgtaactgc tgctgggatt acacatggca tggatgagct ctacaaaagc
      721 ggttccggac cggatcaaac aagtttgtac aaaaaagttg gcggccgcac catgaaaggc
      781 aagttaacag gtaaagacaa attaatagtg gatgagtttg aagaactggg agaacaggaa
      841 tccgatatcg atgaatttga tcttcttgaa ggtgatgaga aattgcctgg cgactcagag
      901 cttgacaaaa ctacatctat ttatcctccc gaaaccagct gggaagtgaa taaagggatg
      961 aacagttctc gtttgcataa actatacagt ttattctttg acaaaagttc cgccttttat
     1021 cttggcgatg atgttagtgt attggaagac aagcctttaa caggtgccta tggttttcag
     1081 agtaaaaaaa atgatcaaca aattttttta ttcagaccgg attctgatta tgttgcagga
     1141 tatcacgttg atgcaaaatc agacgctgga tgggtaaatg acaaactcga tcgtcgttta
     1201 tctgagatta gtgaattttg tagtaaggcc actcaacctg caacgtttat acttcctttt
     1261 gttgaaatgc ccactgacat taccaaaggg gttcaacacc aggtattgtt aactattagc
     1321 tacgatccaa aaagtaaaca actcactccc actgtctatg actccattgg acgagatacc
     1381 tactctgaat cgttatcatc ctattttaaa ggcaaatata gaacaacttg cgatgaaatt
     1441 ttgacccaaa gtattgaaaa agctattaag agcactgatt ttactcttgg taaatttaca
     1501 cgagctgctt ataaccatca aaaccgcctc accgaaggca atgcggggtc ttataccttc
     1561 aggacgatta aagaggttat ttcatcctca gcacaaggta ctgaagtaaa aattcctggc
     1621 tcaggatata ttacttctaa cagttattta acaagtcagc acgtacaaga cattgaaagt
     1681 tgcattaaat atagaaattt gggggttgtt gatatcgaaa gtgctttgac tgaaggtaaa
     1741 acattaccgg tacagctttc tgaatttatt gttgccttgg aagattatgg gaaattacga
     1801 agtcaacaat ctgaaaaaag catgttgaat tttattggct acagcaagac agcaaaattg
     1861 accgctgttg agctgctaat aggcatttta aatgatatca aaggcaaaaa tgaaatcagt
     1921 gaatctcaat atgataaatt agtaaaggaa gtcgattgtt taatggattc atctttagga
     1981 aagttggtac aatttcacct taaaaattta ggcgctgaat cactacaaaa actggtactt
     2041 ccgtgtgtca agtttgatga tacgatagat gattttgtga ctatcgaaaa agacgaacta
     2101 ttcgatgtac ccgatattac aggggaagag cttgcctcta aaaaagggat tgagcaaggt
     2161 gctctggata aagaggcttt actaaaacaa aaacagatta aaacggattt gttagacctt
     2221 cgcgaagaag ataaaaccgg tctgaaaaaa ccattgcatg gtggcattaa ggtaaaatag
     2281 ttaattaatt acccaacttt cttgtacaaa gtggtgataa ttaattaaga taaacccgct
     2341 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2401 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2461 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2521 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     2581 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     2641 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2701 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2761 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2821 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2881 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2941 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3001 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     3061 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3121 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3181 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3241 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3301 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     3361 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     3421 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3481 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3541 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3601 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3661 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3721 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3781 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3841 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3901 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3961 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     4021 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     4081 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4141 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4201 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     4261 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     4321 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4381 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4441 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4501 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4561 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4621 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4681 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4801 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4861 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4921 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4981 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5041 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5101 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5161 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5221 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5281 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5341 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5401 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5461 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5521 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5581 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5641 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5701 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5761 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5821 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5881 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5941 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6001 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6061 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6121 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6181 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6241 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6301 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6361 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6421 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6481 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6541 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6601 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6661 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6721 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     6781 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     6841 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     6901 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     6961 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     7021 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     7081 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     7141 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     7201 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     7261 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     7321 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     7381 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     7441 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     7501 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     7561 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
     7621 atacgactca ctatagggag acccaagctg gctagacacc
//
