LOCUS       sequence-292250-        6092 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 6092)
  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 6092)
  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..6092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        22..401
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        402..605
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     556..576
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     602..626
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        836..854
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     836..853
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     867..886
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             932..1585
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1543..1565
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             join(1601..1603,1604..1606,1607..2278)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="MVRSSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRH
                     HLFL"
     primer_bind     complement(1734..1752)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1784..1804)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2002..2021
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2196..2219
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     regulatory      2400..2409
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2413..2547
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2450..2469)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2504..2523
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2616..2973
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2616..2807
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2639..2659)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2824..2959
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2993..3010)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2993..3009)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3002..3024)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3222..3677
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3309..3328)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3519..3540
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3694..3713)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3813..3835
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3873..3891)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3959..4063
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4064..4132,4133..4924)
                     /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(4282..4301)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5095..5683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5584..5603
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5837..5854
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5971..5992
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6007..6012,6013..6030,6031..6037)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6045..6061
                     /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     6050..6072
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6069..6085
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6069..6085
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cgaattaatt cgagctcgcc cgacattgat tattgactag ttattaatag taatcaatta
       61 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      121 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      181 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      241 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      301 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      361 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      421 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      481 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      541 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      601 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
      661 atagaagaca ccgggaccga tccagcctcc gcggccggga acggtgcatt ggaacgcgga
      721 ttccccgtgc caagagtgac gtaagtaccg cctatagagt ctataggccc acccccttgg
      781 cttcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacgattta
      841 ggtgacacta tagaataaca tccactttgc ctttctctcc acaggtgtcc actcccaggt
      901 ccaactgcac ctcggttcta tcgagccagc catgtcccct atactaggtt attggaaaat
      961 taagggcctt gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga
     1021 gcatttgtat gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt
     1081 ggagtttccc aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc
     1141 catcatacgt tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc
     1201 agagatttca atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc
     1261 atatagtaaa gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct
     1321 gaaaatgttc gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca
     1381 tcctgacttc atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct
     1441 ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga
     1501 taagtacttg aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt
     1561 tggtggtggc gaccatcctc caaaatcgga tggcggccgc atggtgcgct cctccgagga
     1621 cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag ggctccgtga acggccacga
     1681 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     1741 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcca
     1801 gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca agaagctgtc
     1861 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     1921 cgtgacccag gactcctccc tgcaggacgg ctccttcatc tacaaggtga agttcatcgg
     1981 cgtgaacttc ccctccgacg gccccgtaat gcagaagaag actatgggct gggaggcctc
     2041 caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca aggccctgaa
     2101 gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg ccaagaagcc
     2161 cgtgcagctg cccggctact actacgtgga ctccaagctg gacatcacct cccacaacga
     2221 ggactacacc atcgtggagc agtacgagcg cgccgagggc cgccaccacc tgttcctgtc
     2281 tagaatcacc accgtggagt ccaactcctc ctggtggacc aactgggtga tccccgccat
     2341 ctccgccctg gtggtggccc tgatgtaccg cctgtacatg gccgaggact agaagcttgg
     2401 ccgccatggc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2461 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2521 atcaatgtat cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa
     2581 ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg
     2641 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2701 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2761 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2821 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2881 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     2941 ggaggctttt ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg
     3001 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     3061 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     3121 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     3181 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     3241 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3301 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3361 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3421 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3481 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3541 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
     3601 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     3661 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3721 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3781 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3841 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     3901 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     3961 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4021 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4081 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4141 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4201 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4261 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4321 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4381 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4441 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4501 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     4561 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     4621 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     4681 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     4741 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     4801 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     4861 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     4921 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     4981 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5041 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5101 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5161 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5221 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5281 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5341 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5401 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5461 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5521 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5581 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5641 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5701 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5761 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     5821 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     5881 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     5941 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     6001 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     6061 atttcacaca ggaaacagct atgacatgat ta
//
