LOCUS       sequence-265989-        6086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targets red flourescent protein (RFP) to the endoplasmic reticulum.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6086)
  AUTHORS   Stefely JA, Zhang Y, Freiberger EC, Kwiecien NW, Thomas HE, Davis 
            AM, Lowry ND, Vincent CE, Shishkova E, Clark NA, Medvedovic M, Coon 
            JJ, Pagliarini DJ, Mercer CA
  TITLE     Mass spectrometry proteomics reveals a function for mammalian 
            CALCOCO1 in MTOR-regulated selective autophagy.
  JOURNAL   Autophagy. 2020 Jan 23. doi: 10.1080/15548627.2020.1719746.
  PUBMED    31971854
REFERENCE   2  (bases 1 to 6086)
  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: "Autophagy. 
            2020 Jan 23. doi: 10.1080/15548627.2020.1719746."
COMMENT     Sequence Label: GST-RFP-cb5
FEATURES             Location/Qualifiers
     source          1..6086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             918..1571
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRAKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1529..1551
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             join(1587..1589,1590..1592,1593..2264)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
                     HLFL"
     primer_bind     complement(1770..1790)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1988..2007
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2182..2205
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     regulatory      2386..2395
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2399..2533
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2436..2455)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2490..2509
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2602..2959
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2602..2793
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2625..2645)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2810..2945
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2979..2996)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2979..2995)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2988..3010)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3208..3663
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3295..3314)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3505..3526
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3680..3699)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3799..3821
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3859..3877)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3945..4049
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4050..4118,4119..4910)
                     /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     complement(4268..4287)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5081..5669
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5570..5589
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5823..5840
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5957..5978
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5993..5998,5999..6016,6017..6023)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6031..6047
                     /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     6036..6058
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6055..6071
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6055..6071
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccacttccca ggtccaactg
      901 tacctcgcga gccagccatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      961 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
     1021 gcgatgaagg tgataaatgg cgagccaaaa agtttgaatt gggtttggag tttcccaatc
     1081 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
     1141 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
     1201 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
     1261 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
     1321 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
     1381 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
     1441 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
     1501 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
     1561 atcctccaaa atcggatggc ggccgcatgg tgcgctcctc caagaacgtc atcaaggagt
     1621 tcatgcgctt caaggtgcgc atggagggca ccgtgaacgg ccacgagttc gagatcgagg
     1681 gcgagggcga gggccgcccc tacgagggcc acaacaccgt gaagctgaag gtgaccaagg
     1741 gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttccagtac ggctccaagg
     1801 tgtacgtgaa gcaccccgcc gacatccccg actacaagaa gctgtccttc cccgagggct
     1861 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     1921 cctccctgca ggacggctgc ttcatctaca aggtgaagtt catcggcgtg aacttcccct
     1981 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctccacc gagcgcctgt
     2041 acccccgcga cggcgtgctg aagggcgaga tccacaaggc cctgaagctg aaggacggcg
     2101 gccactacct ggtggagttc aagtccatct acatggccaa gaagcccgtg cagctgcccg
     2161 gctactacta cgtggactcc aagctggaca tcacctccca caacgaggac tacaccatcg
     2221 tggagcagta cgagcgcacc gagggccgcc accacctgtt cctgtctaga atcactactg
     2281 tcgagtctaa ttccagttgg tggaccaact gggtgatccc agccatctca gccctggtgg
     2341 tagctctgat gtatcgcctc tacatggcag aagattagaa gcttggccgc catggcccaa
     2401 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2461 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2521 tcatgtctgg atcgggaatt aattcggcgc agcaccatgg cctgaaataa cctctgaaag
     2581 aggaacttgg ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     2641 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2701 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2761 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     2821 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     2881 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     2941 aggcctaggc ttttgcaaaa agctgttaac agcttggcac tggccgtcgt tttacaacgt
     3001 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     3061 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     3121 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3181 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     3241 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3301 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3361 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3421 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3481 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3541 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3601 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3661 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     3721 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3781 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3841 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     3901 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3961 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4021 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4081 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4141 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4201 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4261 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4321 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4381 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4441 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4501 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     4561 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     4621 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     4681 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     4741 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     4801 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4861 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4921 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4981 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5041 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5101 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5161 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5221 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5281 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5341 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5401 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5461 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5521 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5581 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5641 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5701 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5761 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5821 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     5881 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     5941 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     6001 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     6061 acagctatga catgattacg aattaa
//
