LOCUS       sequence-403983-        5695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses HA-GPR35 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5695)
  AUTHORS   Wyant GA, Yu W, Doulamis IP, Nomoto RS, Saeed MY, Duignan T, McCully
            JD, Kaelin WG Jr
  TITLE     Mitochondrial remodeling and ischemic protection by G 
            protein-coupled receptor 35 agonists.
  JOURNAL   Science. 2022 Aug 5;377(6606):621-629. doi: 10.1126/science.abm1638.
            Epub 2022 Aug 4.
  PUBMED    35926043
REFERENCE   2  (bases 1 to 5695)
  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"; doi: 
            "10.1126/science.abm1638"; journalName: "Science"; date: "2022-08-5-
            5"; volume: "377"; issue: "6606"; pages: "621-629"
COMMENT     Sequence Label: pRK5-HA-GPR35
FEATURES             Location/Qualifiers
     source          1..5695
                     /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"
     regulatory      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1995..2004
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2008..2142
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2045..2064)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2099..2118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2211..2568
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2211..2402
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2234..2254)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2419..2554
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2588..2605)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2588..2604)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2597..2619)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2817..3272
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2904..2923)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3114..3135
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3289..3308)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3408..3430
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3468..3486)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3554..3658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3659..3727,3728..4519)
                     /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"
     rep_origin      4690..5278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5179..5198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5432..5449
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5566..5587
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5602..5607,5608..5625,5626..5632)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5640..5656
                     /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     5645..5667
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5664..5680
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5664..5680
                     /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 ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcccgaac cgacagtcgg tctcttcacc aaggccattc
      961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
     1021 gtgggtcgac gatgaatggc acctacaaca cctgtggctc cagcgacctc acctggcccc
     1081 cagcgatcaa gctgggcttc tacgcctact tgggcgtcct gctggtgcta ggcctgctgc
     1141 tcaacagcct ggcgctctgg gtgttctgct gccgcatgca gcagtggacg gagacccgca
     1201 tctacatgac caacctggcg gtggccgacc tctgcctgct gtgcaccttg cccttcgtgc
     1261 tgcactccct gcgagacacc tcagacacgc cgctgtgcca gctctcccag ggcatctacc
     1321 tgaccaacag gtacatgagc atcagcctgg tcacggccat cgccgtggac cgctatgtgg
     1381 ccgtgcggca cccgctgcgt gcccgcgggc tgcggtcccc caggcaggct gcggccgtgt
     1441 gcgcggtcct ctgggtgctg gtcatcggct ccctggtggc tcgctggctc ctggggattc
     1501 aggagggcgg cttctgcttc aggagcaccc ggcacaattt caactccatg gcgttcccgc
     1561 tgctgggatt ctacctgccc ctggccgtgg tggtcttctg ctccctgaag gtggtgactg
     1621 ccctggccca gaggccaccc accgacgtgg ggcaggcaga ggccacccgc aaggctgccc
     1681 gcatggtctg ggccaacctc ctggtgttcg tggtctgctt cctgcccctg cacgtggggc
     1741 tgacagtgcg cctcgcagtg ggctggaacg cctgtgccct cctggagacg atccgtcgcg
     1801 ccctgtacat aaccagcaag ctctcagatg ccaactgctg cctggacgcc atctgctact
     1861 actacatggc caaggagttc caggaggcgt ctgcactggc cgtggctccc cgtgctaagg
     1921 cccacaaaag ccaggactct ctgtgcgtga ccctcgccta agcggccgct aagtaagtaa
     1981 ggatccccag cttggccgcc atggcccaac ttgtttattg cagcttataa tggttacaaa
     2041 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2101 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcgggaatta attcggcgca
     2161 gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga
     2221 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2281 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2341 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2401 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2461 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     2521 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctgttaaca
     2581 gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     2641 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     2701 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt
     2761 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     2821 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2881 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2941 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3001 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     3061 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3121 cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg
     3181 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3241 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     3301 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3361 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3421 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     3481 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     3541 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3601 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3661 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3721 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3781 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3841 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3901 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3961 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4021 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4081 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4141 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4201 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4261 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4321 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     4381 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4441 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4501 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4561 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4621 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4681 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4741 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4801 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4861 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4921 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4981 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5041 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5101 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5161 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5221 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5281 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5341 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5401 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     5461 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     5521 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     5581 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     5641 tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga attaa
//
