LOCUS       sequence-396890-        6344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mutated Translocon-associated protein 
            subunit alpha.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6344)
  AUTHORS   Karki S, Javanainen M, Rehan S, Tranter D, Kellosalo J, Huiskonen 
            JT, Happonen L, Paavilainen V
  TITLE     Molecular view of ER membrane remodeling by the Sec61/TRAP 
            translocon.
  JOURNAL   EMBO Rep. 2023 Dec 6;24(12):e57910. doi: 10.15252/embr.202357910. 
            Epub 2023 Nov 20.
  PUBMED    37983950
REFERENCE   2  (bases 1 to 6344)
  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: "EMBO Rep.";
            date: "2023-12-6"; volume: "24(12):e57910. doi"; pages: " 
            10.15252/embr.202357910. Epub 2023 Nov 20"
COMMENT     Sequence Label: TRAPa(E162A,M164A,R167A)-3xFLAG-pCDNA
FEATURES             Location/Qualifiers
     source          1..6344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     449..468
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(687..791)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(866..885)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1057..1436
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1437..1640
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1591..1611
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1685..1704
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1685..1703
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2644..2709
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2757..2774)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2763..2987
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3033..3461
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3120..3139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3330..3351
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3470..3490)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3475..3804
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3655..3790
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3717..3736
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3871..4665
                     /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(3925..3944)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4535..4554
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4839..4960
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4876..4895)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4930..4949
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5009..5025)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5009..5025)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5022..5044)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5033..5049
                     /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(5057..5063,5064..5081,5082..5087))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5102..5123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5240..5257)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5411..5999)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5491..5510)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6170..6344,1..617,618..686))
                     /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"
ORIGIN
        1 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
       61 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
      121 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
      181 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
      241 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
      301 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
      361 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
      421 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
      481 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
      541 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
      601 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
      661 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
      721 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
      781 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
      841 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
      901 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
      961 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     1021 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     1081 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1141 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     1201 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1261 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1321 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1381 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     1441 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     1501 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     1561 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     1621 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg
     1681 aaattaatac gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttgg
     1741 taccgagctc ggatccacta gtccagtgtg gtggaattcg ccaccatgag actcctcccc
     1801 cgcttgctgc tgcttctctt actcgtgttc cctgccactg tcttgttccg aggcggcccc
     1861 agaggcttgt tagcagtggc acaagatctt acagaggatg aagaaacagt agaagattcc
     1921 ataattgagg atgaagatga tgaagccgag gtagaagaag atgaacccac agatttggta
     1981 gaagataaag aggaagaaga tgtgtctggt gaacctgaag cttcaccgag tgcagataca
     2041 actatactgt ttgtaaaagg agaagatttt ccagcaaata acattgtgaa gttcctggta
     2101 ggctttacca acaagggtac agaagatttt attgttgaat ccttagatgc ctcattccgt
     2161 tatcctcagg actaccagtt ttatatccag aatttcacag ctcttcctct gaacactgta
     2221 gtgccacccc agagacaggc aacttttgag tactctttca ttcctgcagc gcccgcgggc
     2281 ggagcaccat ttggtttggt catcaatctg aactacaaag atttgaacgg caatgtattc
     2341 caagatgcag tcttcaatca aacagttaca gttattgaaa gagaggatgg gttagatgga
     2401 gaaacaatct ttatgtatat gttccttgct ggtcttgggc ttctggttat tgttggcctt
     2461 catcaactcc tagaatctag aaagcgtaag agacccatac agaaagtaga aatgggtaca
     2521 tcaagtcaga atgatgttga catgagttgg attcctcagg aaacattgaa tcaaatcaat
     2581 aaagcttcac caagaaggtt gcccaggaaa cgggcacaga agagatcagt gggatctgat
     2641 gaggactata aagatcatga cggtgattac aaggatcacg atatcgacta taaagatgac
     2701 gacgacaagt aggcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     2761 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2821 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2881 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2941 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3001 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3061 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3121 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3181 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3241 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3301 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3361 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3421 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3481 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3541 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3601 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3661 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3721 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3781 tttggaggcc taggcttttg caaaaagctc ccgggagctt atatatccat tttcggatct
     3841 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     3901 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3961 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4021 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4081 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4141 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4201 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4261 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4321 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4381 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4441 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4501 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4561 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4621 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4681 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4741 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4801 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4861 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4921 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4981 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5041 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5101 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5161 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5221 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5281 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5341 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5401 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5461 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5521 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5581 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5641 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5701 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5761 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5821 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5881 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5941 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6001 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6061 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6121 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6181 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6241 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6301 tgataccgcg agacccacgc tcaccggctc cagatttatc agca
//
