LOCUS       sequence-307458-        7817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Split Fluorescent Reporter for ER Retrograde Trafficking.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7817)
  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 7817)
  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: ER-HA-mNG21-10
FEATURES             Location/Qualifiers
     source          1..7817
                     /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             2515..2541
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2803..2812
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    3226..3793
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3243..3795
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3387..3404)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3614..3633
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3795..4193
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    4207..4795
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4260..4280)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4678..4689)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4870..5103
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5222..5240)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5308..5412
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5413..5481,5482..6273)
                     /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(5631..5650)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6444..7032
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6933..6952
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7186..7203
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7443..7464
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7479..7484,7485..7502,7503..7509)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7517..7533
                     /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     7522..7544
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7541..7557
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7541..7557
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7607..7626
                     /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 ccgcatggga
     2461 tggagctgta tcatcctctt cttggtagca acagctacag gcgcgcactc cggataccca
     2521 tacgatgttc cagattacgc tggaggtatg gtgagcaagg gtgaggagga taacatggcc
     2581 tctctcccag cgactcatga gttacacatc tttggctcca tcaacggtgt ggactttgac
     2641 atggtgggtc agggtaccgg caatccaaat gatggttatg aggagttaaa cctgaagtcc
     2701 accaagggtg acctccagtt ctccccctgg attctggtcc ctcatatcgg gtatggcttc
     2761 catcagtacc tgccctaccc tgacgggatg tcgcctttcc aggccgccat ggtagatggc
     2821 tccggatacc aagtccatcg cacaatgcag tttgaagatg gtgcctccct tactgttaac
     2881 taccgctaca cctacgaggg aagccacatc aaaggagagg cccaggtgat ggggactggt
     2941 ttccctgctg acggtcctgt gatgaccaac acgctgaccg ctgcggactg gtgcatgtcg
     3001 aagaagactt accccaacga caaaaccatc atcagtacct ttaagtggag ttacaccact
     3061 gtaaatggca aacgctaccg gagcactgcg cggaccacct acacctttgc caagccaatg
     3121 gcggctaact atctgaagaa ccagccgatg tacgtgttcc gtaagacgga gctcaagcac
     3181 tccatgggag agaaggatga actgtaatag ctcgagtcta gaggatccct cccccccccc
     3241 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     3301 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     3361 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     3421 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     3481 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     3541 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     3601 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     3661 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     3721 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
     3781 aacacgatga taatatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag
     3841 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc
     3901 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt
     3961 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta
     4021 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg
     4081 tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga
     4141 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taaatcgata
     4201 gatcctaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     4261 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     4321 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     4381 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     4441 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     4501 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     4561 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     4621 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4681 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4741 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgag
     4801 atcctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     4861 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
     4921 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     4981 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     5041 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     5101 gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     5161 cagagagtga gaggcccggg ttaattaagg aaagggctag atcattcttg aagacgaaag
     5221 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5281 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5341 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5401 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5461 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5521 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5581 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5641 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5701 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5761 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5821 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5881 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5941 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6001 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6061 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6121 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6181 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6241 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6301 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6361 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6421 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6481 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6541 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6601 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6661 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6721 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6781 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6841 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6901 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6961 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7021 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7081 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     7141 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     7201 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     7261 taatgcagca agctcatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     7321 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     7381 aaaagctccc cgtggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7441 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     7501 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg
     7561 attacgaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     7621 ctcatcaatg tatcttatca tgtctggatc aactggataa ctcaagctaa ccaaaatcat
     7681 cccaaacttc ccaccccata ccctattacc actgccaatt acctgtggtt tcatttactc
     7741 taaacctgtg attcctctga attattttca ttttaaagaa attgtatttg ttaaatatgt
     7801 actacaaact tagtagt
//
