LOCUS       sequence-409429-        8228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Truncated form of ALIX that lacks C-terminal proline-rich domain; 
            C-terminally tagged with mNeonGreen..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8228)
  AUTHORS   Elias RD, Zhu Y, Su Q, Ghirlando R, Zhang J, Deshmukh L
  TITLE     Reversible phase separation of ESCRT protein ALIX through tyrosine 
            phosphorylation.
  JOURNAL   Sci Adv. 2023 Jul 14;9(28):eadg3913. doi: 10.1126/sciadv.adg3913. 
            Epub 2023 Jul 14.
  PUBMED    37450591
REFERENCE   2  (bases 1 to 8228)
  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: "Sci Adv."; 
            date: "2023-07-14"; volume: "9(28):eadg3913. doi"; pages: " 
            10.1126/sciadv.adg3913. Epub 2023 Jul 14"
COMMENT     Sequence Label: ALIX(1-702)-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..8228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        139..342
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     293..313
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     387..406
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        387..405
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             444..461
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2841..2850
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3343..3360)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3349..3573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3619..4047
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3706..3725)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3916..3937
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4056..4076)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4061..4390
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4241..4376
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4303..4322
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4457..5251
                     /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(4511..4530)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5121..5140
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5425..5546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5462..5481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5516..5535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5595..5611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5595..5611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5608..5630)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5619..5635
                     /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(5643..5649,5650..5667,5668..5673))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5688..5709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5826..5843)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5997..6585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6077..6096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6756..7547,7548..7616))
                     /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     7379..7398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7617..7721)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7796..7815)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7987..8228,1..138)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
       61 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      121 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      181 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      241 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      301 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa
      361 cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctggc
      421 tagcgtttaa acttaagctt atgcatcacc atcaccatca tggcagcggc agcatggcga
      481 cctttattag cgtgcagctg aaaaaaacga gcgaagtgga tctggcgaaa ccgctggtga
      541 aatttattca gcagacctat ccgagcggcg gcgaagaaca agcgcagtat tgtcgcgcag
      601 ctgaagagct gagcaaattg cgccgcgccg cggttggtcg cccgctggat aaacatgaag
      661 gcgcgctgga aaccctgctg cgctattatg atcagatttg cagcattgaa ccgaaatttc
      721 cgtttagcga aaatcagatt tgcctgacct ttacctggaa agatgcgttt gataaaggca
      781 gcctgtttgg cggcagcgtg aaactggctc tggctagcct gggctatgaa aaaagctgcg
      841 tgctgtttaa ctgcgcggcc ctggcgagtc agattgcggc ggaacagaac ctggataacg
      901 atgaaggcct gaaaattgca gcgaagcatt accaatttgc gagcggcgcg tttctgcata
      961 ttaaagaaac cgtgctgagc gcgctgagcc gcgaaccgac cgtggatatt agcccggata
     1021 ccgtgggcac cctgagcctg attatgctgg cgcaagcaca agaagtgttt tttctgaaag
     1081 cgacccgcga taaaatgaaa gatgcgatta ttgcgaaact ggcgaaccaa gcggcggatt
     1141 attttggcga tgcgtttaaa cagtgtcagt ataaagatac cctgccgaaa gaagtgtttc
     1201 cggtgctggc ggcgaaacat tgtataatgc aagcgaacgc ggaatatcac caaagtatct
     1261 tggcgaagca gcagaaaaaa tttggcgaag aaattgcgcg cctgcagcat gcggcggaac
     1321 tgattaaaac cgtggcgagc cgctatgatg aatatgtgaa cgtgaaagat tttagcgata
     1381 aaattaaccg cgcgctggcg gcggcgaaaa aagataacga ttttatttat catgatcgcg
     1441 tgccggatct gaaagacctg gacccgattg gcaaagcgac cctggtgaaa agcaccccgg
     1501 tgaacgtgcc gattagtcag aaatttaccg atctgtttga aaaaatggtg ccggtgagcg
     1561 tgcagcagag cctggcggcg tataatcagc gcaaagcgga tctggtgaac cgcagcattg
     1621 cgcagatgcg cgaagcgacc accctggcga acggcgtgct ggcgagcctg aacctgccgg
     1681 cggcgattga agatgtgagc ggcgataccg tgccgcaatc tatcctgacc aaaagccgca
     1741 gcgtgattga acaaggcggc attcagaccg tggatcagct gattaaagaa ctgccggaac
     1801 tgctgcaacg caatcgcgaa attctggatg aaagcctgcg cctgctggat gaagaagaag
     1861 cgaccgataa cgatctgcgc gcgaaattta aagaacgctg gcagcgcacc ccgagcaacg
     1921 aactgtataa accgctgcgc gcggaaggca ccaactttcg caccgtgctg gataaagcgg
     1981 tgcaagcgga tggccaagtg aaagaatgct atcagagcca tcgcgatacc attgtgctgc
     2041 tgtgcaaacc ggaaccggaa ctgaacgcgg cgattccgag cgcgaacccg gcgaaaacca
     2101 tgcaaggcag cgaagtggtg aacgtgctga aaagcctgct gagcaacctg gatgaagtga
     2161 aaaaagaacg cgaaggcctg gaaaacgatc tgaaaagcgt gaactttgat atgacgagca
     2221 aatttctgac cgcgctggcg caagatggcg tgattaacga agaagcgctg agcgtgaccg
     2281 aactggatcg cgtgtatggc ggcctgacca ccaaagtgca agaaagcctg aaaaaacaag
     2341 aaggcctgct gaaaaacatt caagtgagcc atcaagaatt tagcaaaatg aaacagagca
     2401 acaacgaagc gaacctgcgc gaagaagtgc tgaaaaacct ggcgaccgcg tatgataact
     2461 ttgtggaact ggtggcgaac ctgaaagaag gcaccaaatt ttataacgaa ctgaccgaaa
     2521 ttctggtgcg ctttcagaac aaatgcagcg atattgtgtt tgcgcgcaaa accgaacgcg
     2581 gatccatggt gagcaagggc gaggaggata acatggcctc tctcccagcg acacatgagt
     2641 tacacatctt tggctccatc aacggtgtgg actttgacat ggtgggtcag ggcaccggca
     2701 atccaaatga tggttatgag gagttaaacc tgaagtccac caagggtgac ctccagttct
     2761 ccccctggat tctggtccct catatcgggt atggcttcca tcagtacctg ccctaccctg
     2821 acgggatgtc gcctttccag gccgccatgg tagatggctc cggctaccaa gtccatcgca
     2881 caatgcagtt tgaagatggt gcctccctta ctgttaacta ccgctacacc tacgagggaa
     2941 gccacatcaa aggagaggcc caggtgaagg ggactggttt ccctgctgac ggtcctgtga
     3001 tgaccaactc gctgaccgct gcggactggt gcaggtcgaa gaagacttac cccaacgaca
     3061 aaaccatcat cagtaccttt aagtggagtt acaccactgg aaatggcaag cgctaccgga
     3121 gcactgcgcg gaccacctac acctttgcca agccaatggc ggctaactat ctgaagaacc
     3181 agccgatgta cgtgttccgt aagacggagc tcaagcactc caagaccgag ctcaacttca
     3241 aggagtggca aaaggccttt accgatgtga tggaattctg cagatatcca gcacagtggc
     3301 ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     3361 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3421 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3481 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3541 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3601 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3661 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3721 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3781 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3841 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3901 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3961 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4021 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     4081 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4141 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4201 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4261 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4321 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4381 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     4441 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4501 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4561 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4621 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4681 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4741 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4801 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4861 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4921 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4981 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     5041 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     5101 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     5161 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     5221 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5281 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5341 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5401 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     5461 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5521 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     5581 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5641 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5701 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5761 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5821 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5881 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5941 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6001 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6061 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6121 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6181 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6241 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6301 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6361 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6421 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6481 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6541 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6601 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6661 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6721 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6781 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6841 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6901 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6961 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7021 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     7081 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7141 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7201 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7261 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7321 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7381 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7441 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7501 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7561 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7621 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7681 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7741 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     7801 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     7861 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     7921 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     7981 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     8041 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     8101 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     8161 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     8221 tacatcaa
//
