LOCUS       sequence-307471-        8034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Membrane Tethered Split Fluorescent Reporter for ER Retragrade.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8034)
  AUTHORS   Luong P, Li Q, Chen PF, Wrighton PJ, Chang D, Dwyer S, Bayer MT, 
            Snapper SB, Hansen SH, Thiagarajah JT, Goessling W, Lencer WI
  TITLE     A quantitative single-cell assay for retrograde membrane traffic 
            enables rapid detection of defects in cellular organization.
  JOURNAL   Mol Biol Cell. 2019 Nov 27:mbcE19070375. doi: 
            10.1091/mbc.E19-07-0375.
  PUBMED    31774722
REFERENCE   2  (bases 1 to 8034)
  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: "Mol Biol 
            Cell. 2019 Nov 27:mbcE19070375. doi: 10.1091/mbc.E19-07-0375."
COMMENT     Sequence Label: Sec61b-HA-mNG21-10
FEATURES             Location/Qualifiers
     source          1..8034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2236..2447
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2462..2749
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG
                     RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     CDS             2759..2785
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      3041..3050
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    3443..4010
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3460..4012
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3604..3621)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3831..3850
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4012..4410
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    4424..5012
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4477..4497)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4895..4906)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5087..5320
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5439..5457)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5525..5629
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5630..5698,5699..6490)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5848..5867)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6661..7249
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7150..7169
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7403..7420
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7660..7681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7696..7701,7702..7719,7720..7726)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7734..7750
                     /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     7739..7761
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7758..7774
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7758..7774
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7824..7843
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtgct
     2221 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     2281 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     2341 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     2401 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacagcgg ccgcggcgcg
     2461 catgcctggt ccgaccccca gtggcactaa cgtgggatcc tcagggcgct ctcccagcaa
     2521 agcagtggcc gcccgggcgg cgggatccac tgtccggcag aggaaaaatg ccagctgtgg
     2581 gacaaggagt gcaggccgca caacctcggc aggcaccggg gggatgtggc gattctacac
     2641 agaagattca cctgggctca aagttggccc tgttccagta ttggttatga gtcttctgtt
     2701 catcgcttct gtatttatgt tgcacatttg gggcaagtac actcgttcgg gaggaggata
     2761 cccatacgat gttccagatt acgctatggt gagcaagggt gaggaggata acatggcctc
     2821 tctcccagcg actcatgagt tacacatctt tggctccatc aacggtgtgg actttgacat
     2881 ggtgggtcag ggtaccggca atccaaatga tggttatgag gagttaaacc tgaagtccac
     2941 caagggtgac ctccagttct ccccctggat tctggtccct catatcgggt atggcttcca
     3001 tcagtacctg ccctaccctg acgggatgtc gcctttccag gccgccatgg tagatggctc
     3061 cggataccaa gtccatcgca caatgcagtt tgaagatggt gcctccctta ctgttaacta
     3121 ccgctacacc tacgagggaa gccacatcaa aggagaggcc caggtgatgg ggactggttt
     3181 ccctgctgac ggtcctgtga tgaccaacac gctgaccgct gcggactggt gcatgtcgaa
     3241 gaagacttac cccaacgaca aaaccatcat cagtaccttt aagtggagtt acaccactgt
     3301 aaatggcaaa cgctaccgga gcactgcgcg gaccacctac acctttgcca agccaatggc
     3361 ggctaactat ctgaagaacc agccgatgta cgtgttccgt aagacggagc tcaagcactc
     3421 catgtaactc gagtctagag gatccctccc ccccccctaa cgttactggc cgaagccgct
     3481 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
     3541 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
     3601 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
     3661 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
     3721 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     3781 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     3841 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     3901 atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     3961 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaag
     4021 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
     4081 cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
     4141 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
     4201 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
     4261 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
     4321 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
     4381 ctgccctctg gttatgtgtg ggagggctaa atcgatagat cctaatcaac ctctggatta
     4441 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     4501 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     4561 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     4621 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     4681 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     4741 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     4801 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     4861 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     4921 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     4981 gagtcggatc tccctttggg ccgcctcccc gcctgagatc ctttaagacc aatgacttac
     5041 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     5101 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     5161 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     5221 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     5281 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt
     5341 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gcccgggtta
     5401 attaaggaaa gggctagatc attcttgaag acgaaagggc ctcgtgatac gcctattttt
     5461 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     5521 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5581 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5641 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5701 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5761 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5821 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     5881 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5941 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     6001 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     6061 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6121 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6181 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6241 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6301 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6361 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6421 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6481 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6541 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6601 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6661 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6721 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6781 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6841 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6901 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6961 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     7021 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     7081 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     7141 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     7201 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     7261 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     7321 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     7381 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     7441 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcaagc tcatggctga
     7501 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     7561 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctccccgt ggcacgacag
     7621 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     7681 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     7741 cggataacaa tttcacacag gaaacagcta tgacatgatt acgaatttca caaataaagc
     7801 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     7861 ctggatcaac tggataactc aagctaacca aaatcatccc aaacttccca ccccataccc
     7921 tattaccact gccaattacc tgtggtttca tttactctaa acctgtgatt cctctgaatt
     7981 attttcattt taaagaaatt gtatttgtta aatatgtact acaaacttag tagt
//
