LOCUS       sequence-201582-        3655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid carrying FRBx2 with C-terminal HA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3655)
  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 3655)
  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(-)-FRBx2
FEATURES             Location/Qualifiers
     source          1..3655
                     /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"
     regulatory      614..623
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             632..910
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             917..943
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(979..998)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(980..998)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1005..1022)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1005..1021)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1014..1036)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(1162..1617)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1299..1320)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1511..1530
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1644..1748
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1749..1817,1818..2609)
                     /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(1967..1986)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2780..3368
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3269..3288
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3522..3539
                     /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 gactagtggt tcctctgggt cttccggttc
      601 ctcagggtcg tcggccacca tggcttctag aatcctctgg catgagatgt ggcatgaagg
      661 cctggaagag gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt
      721 gctggagccc ttgcatgcta tgatggaacg gggcccccag actctgaagg aaacatcctt
      781 taatcaggcc tatggtcgag atttaatgga ggcccaagag tggtgcagga agtacatgaa
      841 atcagggaat gtcaaggacc tcacccaagc ctgggacctc tattatcatg tgttccgacg
      901 aatctcaaag actagttatc cgtacgacgt accagactac gcataaagcg gccgccaccg
      961 cggtggagct ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta
     1021 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     1081 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     1141 cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     1201 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     1261 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     1321 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     1381 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     1441 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     1501 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     1561 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     1621 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     1681 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     1741 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     1801 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     1861 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     1921 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     1981 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2041 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2101 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2161 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2221 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     2281 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     2341 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     2401 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     2461 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     2521 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     2581 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     2641 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     2701 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2761 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2821 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2881 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     2941 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3001 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3061 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3121 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3181 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3241 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3301 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3361 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3421 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3481 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3541 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     3601 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaat
//
