LOCUS       sequence-409402-        8726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Full-length ALIX with P801G mutation in proline-rich domain; 
            C-terminally tagged with mNeonGreen..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8726)
  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 8726)
  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(P801G)-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..8726
                     /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      3339..3348
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3841..3858)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3847..4071
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4117..4545
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4204..4223)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4414..4435
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4554..4574)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4559..4888
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4739..4874
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4801..4820
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4955..5749
                     /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(5009..5028)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5619..5638
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5923..6044
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5960..5979)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6014..6033
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6093..6109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6093..6109)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6106..6128)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6117..6133
                     /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(6141..6147,6148..6165,6166..6171))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6186..6207
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6324..6341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6495..7083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6575..6594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7254..8045,8046..8114))
                     /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     7877..7896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8115..8219)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8294..8313)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8485..8726,1..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 acgaactgct gaaagatctg cagcagagca ttgcccgcga accaagcgcc ccaagcatcc
     2641 cgacgccagc ctatcagagt agtccggcgg gtggtcatgc gccgacccca ccaacgccgg
     2701 ccccacgtac gatgccgcca accaaaccgc aaccaccagc gcgtccgcca ccaccagttc
     2761 tcccagccaa tcgcgcccca agtgcgaccg cgccaagccc agttggtgcg ggtaccgccg
     2821 cgccagcgcc aagtcaaacg ccgggtagtg ccccaccacc acaagcgcaa ggcggcccat
     2881 atccgacgta cccgggctat ccgggctact gccagatgcc aatgccgatg ggttacaacc
     2941 catacgcgta cggccagtat aacatgccgt acccaccggt gtaccaccag agtccgggcc
     3001 aagccccata cccgggtcca cagcagccaa gctacccgtt cccgcagcca ccacagcaaa
     3061 gttactaccc gcagcaagga tccatggtga gcaagggcga ggaggataac atggcctctc
     3121 tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg
     3181 tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca
     3241 agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc
     3301 agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg
     3361 gctaccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc
     3421 gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc
     3481 ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga
     3541 agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa
     3601 atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg
     3661 ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca
     3721 agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg gaattctgca
     3781 gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag
     3841 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3901 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3961 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     4021 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     4081 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     4141 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4201 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4261 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4321 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4381 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4441 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     4501 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     4561 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4621 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     4681 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     4741 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4801 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     4861 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     4921 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     4981 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     5041 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     5101 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     5161 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     5221 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     5281 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     5341 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     5401 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     5461 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     5521 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     5581 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     5641 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     5701 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     5761 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     5821 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5881 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     5941 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     6001 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     6061 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     6121 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     6181 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6241 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6301 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     6361 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     6421 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     6481 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     6541 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     6601 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     6661 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6721 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6781 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6841 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6901 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6961 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     7021 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     7081 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     7141 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     7201 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     7261 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     7321 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     7381 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     7441 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     7501 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     7561 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     7621 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     7681 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7741 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7801 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7861 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7921 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7981 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     8041 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     8101 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     8161 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     8221 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     8281 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     8341 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     8401 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     8461 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
     8521 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     8581 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     8641 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     8701 aaactgccca cttggcagta catcaa
//
