LOCUS       sequence-442322-        7139 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Synchronize the trafficking of LAP from the ER..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7139)
  AUTHORS   Ecard J, Lian YL, Divoux S, Gouveia Z, Vigne E, Perez F, Boncompain 
            G
  TITLE     Lysosomal membrane proteins LAMP1 and LIMP2 are segregated in the 
            Golgi apparatus independently of their clathrin adaptor binding 
            motif.
  JOURNAL   Mol Biol Cell. 2024 Mar 1;35(3):ar42. doi: 10.1091/mbc.E23-06-0251. 
            Epub 2024 Jan 17.
  PUBMED    38231876
REFERENCE   2  (bases 1 to 7139)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol 
            Cell."; date: "2024-03-1"; volume: "35(3):ar42. doi"; pages: " 
            10.1091/mbc.E23-06-0251. Epub 2024 Jan 17"
COMMENT     Sequence Label: Str-KDEL_SBP-EGFP-LAP
FEATURES             Location/Qualifiers
     source          1..7139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1646..1875
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1858..1877
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    1947..2520
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2114..2131)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2341..2360
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     sig_peptide     2547..2606
                     /codon_start=1
                     /product="human interleukin-2 signal sequence"
                     /label=IL-2 signal sequence
                     /translation="MYRMQLLSCIALSLALVTNS"
     CDS             2607..2717
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             join(2727..2729,2730..3440)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2727..2729,2730..3440)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2769..2790)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3030..3049)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3377..3398
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4759..4880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4796..4815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4982..4998)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4982..4998)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4995..5017)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5006..5022
                     /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(5030..5036,5037..5054,5055..5060))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5075..5096
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5213..5230)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5384..5972)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5464..5483)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6143..6934,6935..7003))
                     /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     6766..6785
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7004..7108)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtatggatg
      961 tatgcgtccg tcttgccctg tggctcctct ggggactcct cctgcaccag ggccagagcc
     1021 tcagccatag tcacagtgag aaggcgacag gaaccagctc gggcgccggt ttaaacgacc
     1081 ctagcaaaga ctcaaaagct caggtgtccg ctgccgaggc tggcattact ggaacatggt
     1141 acaatcagct cgggagcacc tttattgtga ctgctggagc cgatggagcc ctcaccggaa
     1201 catacgaatc tgctgtggga aacgccgaat cacggtacgt cctcactggc cgatacgata
     1261 gtgcccctgc caccgacgga tctgggactg ccctgggatg gactgtcgct tggaaaaaca
     1321 actaccggaa tgctcattct gccacaacat ggagtggaca gtacgtggga ggcgctgagg
     1381 ctagaatcaa tacacagtgg ctgctcacat ctggcacaac cgaggcaaat gcttggaaat
     1441 ccaccctggt gggacatgac acattcacca aagtgaaacc ctccgccgct tcaatcgatg
     1501 ccgccaaaaa agccggagtc aacaacggca atcctctgga tgccgtccag cagctcggac
     1561 cgaaagatga actgtgagcg gccgcataga taactgatcc agtgtgctgg aattaattcg
     1621 ctgtctgcga gggccagctg ttggggtgag tactccctct caaaagcggg catgacttct
     1681 gcgctaagat tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgcggtg
     1741 atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt gttgtcaagc
     1801 ttgaggtgtg gcaggcttga gatctggcca tacacttgag tgacaatgac atccactttg
     1861 cctttctctc cacaggtgtc cactcccagg tccaactgca ggtcgagcat gcatctaggg
     1921 cggccaattc cgcccctctc cccccccccc tttccctccc ccccccctaa cgttactggc
     1981 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     2041 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     2101 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     2161 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     2221 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     2281 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     2341 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     2401 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     2461 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     2521 gcttgccaca acccgggagg cgcgccatgt acaggatgca actcctgtct tgcattgcac
     2581 taagtcttgc acttgtcacg aattccgacg agaagaccac tggttggcga ggtggacacg
     2641 ttgttgaagg actggctggg gaacttgaac aacttcgtgc acgactggag catcacccac
     2701 aaggtcaacg tgaaccacct gcaggtgtga gcaagggcga ggagctgttc accggggtgg
     2761 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2821 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2881 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     2941 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     3001 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     3061 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     3121 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     3181 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     3241 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     3301 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     3361 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     3421 gcatggacga gctgtacaag ggccggccac ggagtctgcg cttcgttacc ttgctgtacc
     3481 gccatggaga ccgttcacca gtgaagacat atcccaagga cccctatcag gaagaagaat
     3541 ggccccaggg gtttggtcag ttaaccaagg aggggatgct acagcactgg gaactgggcc
     3601 aggccctgcg gcagcgctat cacggcttcc taaacacctc ttatcaccgg caagaggttt
     3661 atgtgcgaag cacagacttt gaccggactc tcatgagtgc tgaggccaac ctggctggac
     3721 tcttccctcc caacgggatg cagcgcttca acccgaacat ctcgtggcag cctattcctg
     3781 tgcacactgt gcccatcact gaggacaggc tgctgaagtt cccgttgggc ccatgtcccc
     3841 gttatgagca gctgcagaac gagacccggc agacaccaga gtatcagaat gagagttctc
     3901 ggaatgcaca atttctggac atggtggcca acgagacagg gcttacagac ctgacactgg
     3961 agaccgtctg gaatgtctat gacacactct tctgtgagca aacgcacggg ctgcgcctgc
     4021 cgccctgggc ctcaccccaa accatgcagc gtctcagccg gctaaaggac ttcagcttcc
     4081 gcttcctctt cggaatctac cagcaggcgg agaaggcccg gcttcagggg ggagtcctgc
     4141 tggctcagat aaggaagaac ctgaccctaa tggcgaccac ctcccagctc cccaagctgc
     4201 tggtttactc tgcgcacgac actaccctgg ttgccctgca aatggcactg gatgtctaca
     4261 atggtgaaca agccccctac gcctcctgcc acatatttga actgtaccag gaagattctg
     4321 ggaatttctc agtggagatg tactttcgga acgagagtga caaggccccc tggccgctca
     4381 gcctgcctgg ctgccctcac cgctgcccac tgcaggactt ccttcgcctc acagagcccg
     4441 tcgtgcccaa ggattggcag caggagtgcc agctggcaag cggtcctgca gacacagagg
     4501 tgattgtggc cttggctgta tgtggctcca tcctcttcct cctcatagtg ctgctcctca
     4561 ccgtcctctt ccggatgcag gcccagcctc ctggctaccg ccacgtcgca gatggggagg
     4621 accacgcctg acttaattaa ctctagataa ctgatcataa tcagccatac cacatttgta
     4681 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     4741 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4801 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4861 aaactcatca atgtatctta acgcgtcgag tgcattctag ttgtggtttg tccaaactca
     4921 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4981 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5041 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5101 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5161 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5221 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5281 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5341 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5401 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5461 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5521 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5581 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5641 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5701 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5761 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5821 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5881 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5941 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6001 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6061 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6121 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6181 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6241 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6301 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6361 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6421 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6481 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6541 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6601 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6661 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6721 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6781 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6841 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6901 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6961 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7021 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7081 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
