LOCUS       sequence-443322-        6305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TMEM9B expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6305)
  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 6305)
  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: pcDNA -TMEM9B-3xHA
FEATURES             Location/Qualifiers
     source          1..6305
                     /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"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1291..2316)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1291..2316)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(2365..2694)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2379..2514
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2433..2452)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2679..2699
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2708..3136)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2818..2839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3030..3049
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3285..3396
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3385..3402
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3445..3525)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     protein_bind    3527..3551
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4146..4170)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4257..4276)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4258..4276)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4321..4524)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4350..4370)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4525..4904
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5076..5095
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5170..5274
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5275..5343,5344..6135)
                     /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(5493..5512)
                     /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 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtagcacgtg ctattccttt gccctcggac gagtgctggg
     1321 gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct
     1381 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca
     1441 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg
     1501 gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg
     1561 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa
     1621 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
     1681 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
     1741 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga
     1801 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc
     1861 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa
     1921 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg
     1981 cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
     2041 ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
     2101 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca
     2161 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc
     2221 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
     2281 ctcgacagac gtcgcggtga gttcaggctt tttcatcacg tgctgatcag atccgaaaat
     2341 ggatatacaa gctcccggga gctttttgca aaagcctagg cctccaaaaa agcctcctca
     2401 ctacttctgg aatagctcag aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat
     2461 tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg
     2521 agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc
     2581 tgctggggag cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg
     2641 catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca
     2701 cagaattaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2761 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2821 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2881 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2941 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3001 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3061 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3121 ccgctacagg gcgcgtgggg atacccccta gagccccagc tggttctttc cgcctcagaa
     3181 gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttccccttg ctgtcctgcc
     3241 ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat
     3301 tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg
     3361 ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag cgggtttaaa
     3421 cgggccctct agactcgagt ttcaagcgta gtctggtaca tcataagggt atgcatagtc
     3481 aggtacgtca taaggataag cgtaatctgg aacatcgtat gggtaaacca ctttgtacaa
     3541 gaaagttggg tagctgagga caacatgccg gtcaaagaca gactttcgct gctcttggac
     3601 ttgaagcttc cagcgctgct gtgcatattc taccttgttc agcacgttgg ctcgactgcg
     3661 ggagcgggct agcacatcgt gtgcatttgc aaaaggctgg tgatccccaa tatcatcatc
     3721 actctgtatc aactgtgcat gtccaaagag gcgcctcttc agtatgggct caaccagagt
     3781 aagatatacc atgtacagaa gtagaaggcc caaaatggag agataaatta taatggtaac
     3841 cttgattgtg acagagcttc tttcttcata tttgcattca cagcgtagac agtatgcttc
     3901 tacatcaggc ccccgcacag gcatgggctc cacaacatga aggcaatcac aatctttctg
     3961 agatatgttc ttattataaa tatgcccaga attttcttta tagggagggc agatacattt
     4021 acatctgaca tcctcgaaat tcttggcggc gtctgacagc tgcgccagca gcagcacgga
     4081 aagcgccagg cacgacaggc tgagcaagga gccaagccga agaaggcctc cccacagggt
     4141 cgccatgcca acttttttgt acaaacttgt tgatatctgc agaattccac cacactggac
     4201 tagtggatcc gagctcggta ccaagcttaa gtttaaacgc tagccagctt gggtctccct
     4261 atagtgagtc gtattaattt cgataagcca gtaagcagtg ggttctctag ttagccagag
     4321 agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc
     4381 ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt
     4441 gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt
     4501 gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg
     4561 ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc
     4621 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg
     4681 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
     4741 tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg
     4801 cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga
     4861 ccccgtaatt gattactatt aataactagt caataatcaa tgtcaacgcg tatatctggc
     4921 ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaattgtc
     4981 ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa
     5041 cacacaagca gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     5101 agtgcaccat aggggatcgg gagatctccc gatccgtcga cgtcaggtgg cacttttcgg
     5161 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5221 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5281 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5341 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5401 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5461 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5521 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5581 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5641 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5701 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5761 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5821 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5881 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5941 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6001 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6061 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6121 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6181 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6241 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6301 tcttc
//
