LOCUS       sequence-356577-        7573 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Co-expresses (via IRES) streptavidin-KDEL and 
            Prosaposin-SBP-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7573)
  AUTHORS   Devireddy S, Ferguson SM
  TITLE     Efficient progranulin exit from the ER requires its interaction with
            prosaposin, a Surf4 cargo.
  JOURNAL   J Cell Biol. 2022 Feb 7;221(2):e202104044. doi: 
            10.1083/jcb.202104044. Epub 2021 Dec 17.
  PUBMED    34919127
REFERENCE   2  (bases 1 to 7573)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2022-02-7"; volume: "221(2):e202104044. doi"; pages: 
            " 10.1083/jcb.202104044. Epub 2021 Dec 17"
COMMENT     Sequence Label: STR-KDEL_PSAP-SBP-mCherry
FEATURES             Location/Qualifiers
     source          1..7573
                     /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    join(1947..2521,2522..2524,2525..2533)
                     /label=IRES2
                     /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"
     CDS             4125..4235
                     /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             4245..4952
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4387..4405)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4655..4674
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    5193..5314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5230..5249)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5416..5432)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5416..5432)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5429..5451)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5440..5456
                     /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(5464..5470,5471..5488,5489..5494))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5509..5530
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5647..5664)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5818..6406)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5898..5917)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6577..7368,7369..7437))
                     /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     7200..7219
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7438..7542)
                     /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 tctccctccc 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 acgccctctt cctcctggcc agcctcctgg
     2581 gcgcggctct agccggcccg gtccttggac tgaaagaatg caccaggggc tcggcagtgt
     2641 ggtgccagaa tgtgaagacg gcgtccgact gcggggcagt gaagcactgc ctgcagaccg
     2701 tttggaacaa gccaacagtg aaatcccttc cctgcgacat atgcaaagac gttgtcaccg
     2761 cagctggtga tatgctgaag gacaatgcca ctgaggagga gatccttgtt tacttggaga
     2821 agacctgtga ctggcttccg aaaccgaaca tgtctgcttc atgcaaggag atagtggact
     2881 cctacctccc tgtcatcctg gacatcatta aaggagaaat gagccgtcct ggggaggtgt
     2941 gctctgctct caacctctgc gagtctctcc agaagcacct agcagagctg aatcaccaga
     3001 agcagctgga gtccaataag atcccagagc tggacatgac tgaggtggtg gcccccttca
     3061 tggccaacat ccctctcctc ctctaccctc aggacggccc ccgcagcaag ccccagccaa
     3121 aggataatgg ggacgtttgc caggactgca ttcagatggt gactgacatc cagactgctg
     3181 tacggaccaa ctccaccttt gtccaggcct tggtggaaca tgtcaaggag gagtgtgacc
     3241 gcctgggccc tggcatggcc gacatatgca agaactatat cagccagtat tctgaaattg
     3301 ctatccagat gatgatgcac atgcaaccca aggagatctg tgcgctggtt gggttctgtg
     3361 atgaggtgaa agagatgccc atgcagactc tggtccccgc caaagtggcc tccaagaatg
     3421 tcatccctgc cctggaactg gtggagccca ttaagaagca cgaggtccca gcaaagtctg
     3481 atgtttactg tgaggtgtgt gaattcctgg tgaaggaggt gaccaagctg attgacaaca
     3541 acaagactga gaaagaaata ctcgacgctt ttgacaaaat gtgctcgaag ctgccgaagt
     3601 ccctgtcgga agagtgccag gaggtggtgg acacgtacgg cagctccatc ctgtccatcc
     3661 tgctggagga ggtcagccct gagctggtgt gcagcatgct gcacctctgc tctggcacgc
     3721 ggctgcctgc actgaccgtt cacgtgactc agccaaagga cggtggcttc tgcgaagtgt
     3781 gcaagaagct ggtgggttat ttggatcgca acctggagaa aaacagcacc aagcaggaga
     3841 tcctggctgc tcttgagaaa ggctgcagct tcctgccaga cccttaccag aagcagtgtg
     3901 atcagtttgt ggcagagtac gagcccgtgc tgatcgagat cctggtggag gtgatggatc
     3961 cttccttcgt gtgcttgaaa attggagcct gcccctcggc ccataagccc ttgttgggaa
     4021 ctgagaagtg tatatggggc ccaagctact ggtgccagaa cacagagaca gcagcccagt
     4081 gcaatgctgt cgagcattgc aaacgccatg tgtggaacgc cgtcgacgag aagaccactg
     4141 gttggcgagg tggacacgtt gttgaaggac tggctgggga acttgaacaa cttcgtgcac
     4201 gactggagca tcacccacaa ggtcaacgtg aaccacctgc aggtgtgagc aagggcgagg
     4261 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     4321 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     4381 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     4441 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     4501 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     4561 gcggcgtggt gaccgtgacc caggactcct ccctacagga cggcgagttc atctacaagg
     4621 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     4681 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     4741 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     4801 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     4861 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     4921 ccaccggtgg catggacgag ctgtacaagt agggccggcc actggaaaat ggcggaacct
     4981 ctctgtctga aaaaactgtg ctgctgctgg tgactccctt tctggccgct gcttggtccc
     5041 tccacccatg aggcctcgag ggccaattaa ttaactctag ataactgatc ataatcagcc
     5101 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     5161 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     5221 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5281 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt cgagtgcatt ctagttgtgg
     5341 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     5401 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5461 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5521 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5581 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5641 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5701 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5761 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5821 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5881 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5941 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6001 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6061 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6121 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6181 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6241 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6301 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6361 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6421 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6481 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6541 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6601 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6661 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6721 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6781 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6841 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6901 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6961 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7021 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7081 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7141 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7201 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7261 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7321 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7381 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7441 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7501 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7561 gccacctgac gtc
//
