LOCUS       sequence-443326-        4591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TMEM230triplemut expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4591)
  AUTHORS   Gonzalez-Lozano MA, Schmid EW, Miguel Whelan E, Jiang Y, Paulo JA, 
            Walter JC, Harper JW
  TITLE     EndoMAP.v1 charts the structural landscape of human early endosome 
            complexes.
  JOURNAL   Nature. 2025 Jul;643(8070):252-261. doi: 10.1038/s41586-025-09059-y.
            Epub 2025 May 28.
  PUBMED    40437099
REFERENCE   2  (bases 1 to 4591)
  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"; doi: 
            "10.1038/s41586-025-09059-y"; journalName: "Nature"; date: 
            "2025-07"; volume: "643"; issue: "8070"; pages: "252-261"
COMMENT     Sequence Label: pGCS-3xHA-TMEM230triplemut
FEATURES             Location/Qualifiers
     source          1..4591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    1143..1277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1180..1199)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1234..1253
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1307..1331
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1718..1742)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1752..1832)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     promoter        complement(1867..1885)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1868..1885)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(1920..2904)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     rep_origin      2975..3430
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3062..3081)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3272..3293
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3456..3560
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3561..3629,3630..4421)
                     /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     complement(3779..3798)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgctccc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tgggtaccgg gcccaatgca ttggcgccgc ggccgcgaat
     1081 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     1141 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1201 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     1261 cttatcatgt ctggatctac gtaatacgac tcactatagt tctagaacca ctttgtacaa
     1321 gaaagctggg ttcagctatg gggtgggtgc ccggggtcat caaagtctgg aatgtcatca
     1381 taggagtaac cacggtagcc tttggatgca tagtaagcga tgcgcaggtg gtaaaatccg
     1441 ggtaggaaca ccagaatgcc aatgatcagc actggaacgg ccaggtctgc cccccctttg
     1501 ctgatgtagc ctgacagcag gagggagcct ataataatga gaaaggcgcc aatcaaaaac
     1561 agcacagtgg caagtgcgat ggccttataa gggatcttag gaggggtttt cttaaactga
     1621 aggtcaatgc agccatcgtc tgtgctggag agccttgaat atttcacttt actactgggg
     1681 attccagtag ccaggttggt acgggacggc atcatggagc ctgctttttt gtacaaactt
     1741 gttggccggc cagcgtagtc tggtacatca taagggtatg catagtcagg tacgtcataa
     1801 ggataagcgt aatctggaac atcgtatggg tacatggatc ctgcaaaaag aacaagtagc
     1861 ttgtattcta tagtgtcacc taaatcaagc ttgctccgac gtccccaggc agaatggcgg
     1921 ttccctaaac gagcattgct tatatagacc tcccattagg cacgcctacc gcccatttac
     1981 gtcaatggaa cgcccatttg cgtcattgcc cctccccatt gacgtcaatg gggatgtact
     2041 tggcagccat cgcgggccat ttaccgccat tgacgtcaat gggagtactg ccaatgtacc
     2101 ctggcgtact tccaatagta atgtacttgc caagttacta ttaatagata ttgatgtact
     2161 gccaagtggg ccatttaccg tcattgacgt caataggggg cgtgagaacg gatatgaatg
     2221 ggcaatgagc catcccattg acgtcaatgg tgggtggtcc tattgacgtc aatgggcatt
     2281 gagccaggcg ggccatttac cgtaattgac gtcaatgggg gaggcgccat atacgtcaat
     2341 aggaccgccc atatgacgtc aataggaaag accatatata gagaccattg acgtcaatgg
     2401 gggagtggct atgggcggta ttaggaagcc ccatatatgg tatatgggac cgcccattgg
     2461 gaggggctat ctacgtcaat aggaaaaccc atatatggaa tactatatgg catagggcca
     2521 atacatagta ttgaacctgg ccaatagcca tattggcata gggccatatt ggatattgcc
     2581 tatatattga tcctggcata tagccaatat ggccgccatt attggcacca tgccaattca
     2641 atatggcgga cttggcacag ggccaagacc cctcctcaaa cttggcaccg tgccaagtag
     2701 acccctcccc agttggcaca gtgccaggtc cgccatattg aattggcatg gtgccaagtc
     2761 cgccatattg aattggctgg ggtccagatc caccatattg aattggcacg agtccatata
     2821 cgccatattg aattggcaca ggtccagatc caccatattg aattggcatg agtccatata
     2881 cgccatattg aattggcggt cgactggcgt aatagcgaag aggcccgcac cgatcgccct
     2941 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     3001 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3061 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3121 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3181 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3241 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3301 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3361 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3421 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3481 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3541 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3601 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3661 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3721 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3781 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     3841 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3901 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3961 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4021 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4081 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4141 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4201 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4261 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4321 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4381 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4441 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4501 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4561 cagaccccgt agaaaagatc aaaggatctt c
//
