LOCUS       sequence-201580-        3316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid carrying FRB with C-terminal HA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3316)
  AUTHORS   Papanikou E, Day KJ, Austin J, Glick BS
  TITLE     COPI selectively drives maturation of the early Golgi.
  JOURNAL   Elife. 2015 Dec 28;4. pii: e13232. doi: 10.7554/eLife.13232.
  PUBMED    26709839
REFERENCE   2  (bases 1 to 3316)
  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: "Elife."; 
            date: "2015-12-28"; volume: "4. pii: e13232. doi"; pages: " 
            10.7554/eLife.13232"
COMMENT     Sequence Label: pBluescriptKS(-)-FRB
FEATURES             Location/Qualifiers
     source          1..3316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     182..198
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     183..199
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      275..284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             293..571
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             578..604
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(640..659)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(641..659)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(666..683)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(666..682)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(675..697)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(823..1278)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(960..981)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1172..1191
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1305..1409
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1410..1478,1479..2270)
                     /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(1628..1647)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2441..3029
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2930..2949
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3183..3200
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag ctcgaaatta accctcacta aagggaacaa aagctggtac cgggcccccc
      181 ctcgaggtcg acggtatcga taagcttgat atcgaattcc tgcagcccgg gggatccagg
      241 ttcctctggg tcttccggtt cctcagggtc gtcggccacc atggcttcta gaatcctctg
      301 gcatgagatg tggcatgaag gcctggaaga ggcatctcgt ttgtactttg gggaaaggaa
      361 cgtgaaaggc atgtttgagg tgctggagcc cttgcatgct atgatggaac ggggccccca
      421 gactctgaag gaaacatcct ttaatcaggc ctatggtcga gatttaatgg aggcccaaga
      481 gtggtgcagg aagtacatga aatcagggaa tgtcaaggac ctcacccaag cctgggacct
      541 ctattatcat gtgttccgac gaatctcaaa gactagttat ccgtacgacg taccagacta
      601 cgcataaagc ggccgccacc gcggtggagc tccaattcgc cctatagtga gtcgtattac
      661 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
      721 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
      781 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta agcgttaata
      841 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
      901 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
      961 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     1021 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     1081 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     1141 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     1201 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     1261 cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1321 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1381 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1441 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1501 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1561 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1621 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1681 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1741 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1801 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1861 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1921 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     1981 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2041 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2101 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2161 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2221 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2281 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2341 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2401 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2461 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2521 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2581 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2641 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2701 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2761 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2821 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2881 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2941 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3001 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3061 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3121 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3181 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     3241 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     3301 gtgagcgcaa cgcaat
//
