LOCUS       sequence-491592-        5404 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Mammalian expression of mCherry-sspB-Sec61 fusion; for optogenetic 
            control of protein recruitment to the ER.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5404)
  TITLE     Secretome translation shaped by Lysosomes and Lunapark-marked ER 
            Junctions
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5404)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCMV-mCherry-sspB-Sec61
FEATURES             Location/Qualifiers
     source          1..5404
                     /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"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1310..1319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1316..2023
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1461..1479)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1729..1748
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2087..2416
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein"
                     /label=SspB nano
                     /note="dimerizes with iLID at high affinity to create iLID 
                     nano (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNARFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     CDS             2447..2734
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="PGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAGR
                     TTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     polyA_signal    2895..3016
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2932..2951)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2986..3005
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3023..3478)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3160..3181)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3372..3391
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3505..3609
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3611..3968
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3611..3802
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3634..3654)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3819..3954
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4003..4797
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4003..4797
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4057..4076)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4667..4686
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4985..5004)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5029..5076
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
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 taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt
     1321 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     1381 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     1441 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     1501 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     1561 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     1621 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     1681 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     1741 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     1801 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     1861 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     1921 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     1981 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtccggac tcagatctgg
     2041 ctccagcgca ggcattgatc tgagcggcct gaccctgcag gaattcagct ccccgaaacg
     2101 ccctaagctg ctgcgtgaat attacgattg gctggttgat aacagcttta ccccatatct
     2161 ggtggtggat gccacatacc tgggcgtgaa cgtgcccgtg gagtatgtga aagacggtca
     2221 gatcgtgctg aatctgtctg caagtgcgac cggcaacctg caactgacaa atgattttat
     2281 ccagttcaac gcccgcttta agggcgtgtc tcgtgaactg tatatcccga tgggtgccgc
     2341 tctggccatt tacgctcgcg agaacggcga tggtgtgatg ttcgaaccag aagaaatcta
     2401 tgacgagctg aatattggtg gcagcgcatc cggcggaagc ggaagccctg gtccgacccc
     2461 cagtggcact aacgtgggat cctcagggcg ctctcccagc aaagcagtgg ccgcccgggc
     2521 ggcgggatcc actgtccggc agaggaaaaa tgccagctgt gggacaagga gtgcaggccg
     2581 cacaacctcg gcaggcaccg gggggatgtg gcgattctac acagaagatt cacctgggct
     2641 caaagttggc cctgttccag tattggttat gagtcttctg ttcatcgctt ctgtatttat
     2701 gttgcacatt tggggcaagt acactcgttc gtaggaattc tgcagtcgac ggtaccgcgg
     2761 gcccgggatc caccggatct agataactga tcataatcag ccataccaca tttgtagagg
     2821 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     2881 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     2941 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3001 tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta
     3061 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3121 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3181 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3241 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3301 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     3361 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     3421 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca
     3481 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3541 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3601 aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa
     3661 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3721 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3781 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3841 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3901 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3961 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     4021 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     4081 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     4141 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     4201 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     4261 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     4321 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     4381 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4441 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4501 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     4561 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4621 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4681 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4741 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4801 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4861 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4921 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc
     4981 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     5041 gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg
     5101 gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt
     5161 ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg
     5221 gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac
     5281 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5341 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5401 cttc
//
