LOCUS       sequence-443325-        4572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TMEM230 expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4572)
  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 4572)
  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-TMEM230
FEATURES             Location/Qualifiers
     source          1..4572
                     /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(1699..1723)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1733..1813)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     promoter        complement(1848..1866)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1849..1866)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(1901..2885)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     rep_origin      2956..3411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3043..3062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3253..3274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3437..3541
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3542..3610,3611..4402)
                     /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(3760..3779)
                     /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 tcactagtca tcaaagtctg gaatgtcatc ataggagtaa ccacggtagc
     1381 ctttggatgc atagtaagcg atgcgcaggt ggtaaaatcc gggtaggaac accagaatgc
     1441 caatgatcag cactggaacg gcccggtctg cccccccttt gctgatgtag cctgacagca
     1501 ggagggagcc tataataatg agaaaggcgc caatcaaaaa cagcacagtg gcaagtgcga
     1561 tggccttata agggatctta ggaggggttt tcttaaactg aaggtcaatg tagccatcgt
     1621 ctgtgctgga gagccttgaa tatttcactt tactactggg gattccagta gccaggttgg
     1681 tacgggacgg catcatggag cctgcttttt tgtacaaact tgttggccgg ccagcgtagt
     1741 ctggtacatc ataagggtat gcatagtcag gtacgtcata aggataagcg taatctggaa
     1801 catcgtatgg gtacatggat cctgcaaaaa gaacaagtag cttgtattct atagtgtcac
     1861 ctaaatcaag cttgctccga cgtccccagg cagaatggcg gttccctaaa cgagcattgc
     1921 ttatatagac ctcccattag gcacgcctac cgcccattta cgtcaatgga acgcccattt
     1981 gcgtcattgc ccctccccat tgacgtcaat ggggatgtac ttggcagcca tcgcgggcca
     2041 tttaccgcca ttgacgtcaa tgggagtact gccaatgtac cctggcgtac ttccaatagt
     2101 aatgtacttg ccaagttact attaatagat attgatgtac tgccaagtgg gccatttacc
     2161 gtcattgacg tcaatagggg gcgtgagaac ggatatgaat gggcaatgag ccatcccatt
     2221 gacgtcaatg gtgggtggtc ctattgacgt caatgggcat tgagccaggc gggccattta
     2281 ccgtaattga cgtcaatggg ggaggcgcca tatacgtcaa taggaccgcc catatgacgt
     2341 caataggaaa gaccatatat agagaccatt gacgtcaatg ggggagtggc tatgggcggt
     2401 attaggaagc cccatatatg gtatatggga ccgcccattg ggaggggcta tctacgtcaa
     2461 taggaaaacc catatatgga atactatatg gcatagggcc aatacatagt attgaacctg
     2521 gccaatagcc atattggcat agggccatat tggatattgc ctatatattg atcctggcat
     2581 atagccaata tggccgccat tattggcacc atgccaattc aatatggcgg acttggcaca
     2641 gggccaagac ccctcctcaa acttggcacc gtgccaagta gacccctccc cagttggcac
     2701 agtgccaggt ccgccatatt gaattggcat ggtgccaagt ccgccatatt gaattggctg
     2761 gggtccagat ccaccatatt gaattggcac gagtccatat acgccatatt gaattggcac
     2821 aggtccagat ccaccatatt gaattggcat gagtccatat acgccatatt gaattggcgg
     2881 tcgactggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     2941 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3001 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3061 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3121 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3181 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3241 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3301 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3361 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3421 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3481 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3541 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3601 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3661 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3721 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3781 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3841 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3901 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3961 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4021 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4081 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4141 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4201 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4261 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4321 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4381 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4441 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4501 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4561 caaaggatct tc
//
