LOCUS       sequence-495867-        7608 bp    DNA     circular SYN 19-DEC-2025
DEFINITION  Expression vector for secreted human GPR124 HRM and GAIN domains, 
            N-terminal 3xFLAG tag, C-terminal 6xHis tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7608)
  AUTHORS   Vallon M, Yuki K, Nguyen TD, Chang J, Yuan J, Siepe D, Miao Y, 
            Essler M, Noda M, Garcia KC, Kuo CJ
  TITLE     A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific 
            Regulation of Wnt Bioavailability.
  JOURNAL   Cell Rep. 2018 Oct 9;25(2):339-349.e9. doi: 
            10.1016/j.celrep.2018.09.045.
  PUBMED    30304675
REFERENCE   2  (bases 1 to 7608)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 19, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2018.09"; journalName: "Cell Rep"; date: 
            "2018-10-9- 9"; volume: "25"; issue: "2"; pages: "339-349.e9"
COMMENT     Sequence Label: p3xFLAG-CMV-9-GPR124 HRM-GAIN-His
FEATURES             Location/Qualifiers
     source          1..7608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    8..24
                     /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)."
     promoter        complement(join(32..38,39..56,57..62))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    77..98
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(215..232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(386..974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(466..485)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1145..1936,1937..2005))
                     /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     1768..1787
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2006..2110)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2224..2243)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2451..2472
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2735..2757
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2749..2766
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2750..2766
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        2927..3306
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3307..3510
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3461..3481
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3541..3585
                     /codon_start=1
                     /product="leader sequence from mouse preprotrypsin"
                     /label=preprotrypsin leader
                     /translation="MSALLILALVGAAVA"
     CDS             3586..3651
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4930..4947
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    4958..5580
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5157..5176)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(5604..5624)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5622..5920
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             5974..6768
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6028..6047)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6638..6657
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7420..7554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7457..7476)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7511..7530
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7559..7578)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7560..7578)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7592..7608)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7592..7608)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(join(7605..7608,1..19))
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
       61 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
      121 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
      181 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
      241 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      301 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      361 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      421 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      481 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      541 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      601 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      661 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      721 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      781 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      841 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      901 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      961 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1021 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1081 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1141 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1201 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1261 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     1321 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1381 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1441 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1501 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1561 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1621 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1681 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1741 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1801 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1861 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     1921 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     1981 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2041 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2101 gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa
     2161 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2221 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2281 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2341 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2401 gccctttgac gttggagtcc acgttcttta agagtccact attaaagaac gtggactctt
     2461 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2521 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2581 ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg caactgttgg
     2641 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     2701 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     2761 gccagtgcca agctgatcta tacattgaat caatattggc aattagccat attagtcatt
     2821 ggttatatag cataaatcaa tattggctat tggccattgc atacgttgta tctatatcat
     2881 aatatgtaca tttatattgg ctcatgtcca atatgaccgc catgttgaca ttgattattg
     2941 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3001 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     3061 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3121 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3181 ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     3241 tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     3301 accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg
     3361 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     3421 cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt
     3481 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagaa ttaattcacc
     3541 atgtctgcac ttctgatcct agctcttgtt ggagctgcag ttgctgacta caaagaccat
     3601 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa gcttgcggcc
     3661 gcgaattcag tggagatcgt ggtgctggag acctctgcct cctactgccc cgccgagcgt
     3721 gttgccaaca accgcgggga cttcaggtgg ccccgaactc tggctggcat cacagcctac
     3781 cagtcctgcc tgcagtatcc cttcacctca gtgcccctgg gcgggggtgc cccgggcacc
     3841 cgagcctccc gccggtgtga ccgtgccggc cgctgggagc caggggacta ctcccactgt
     3901 ctctacacca acgacatcac cagggtgctg tacaccttcg tgctgatgcc catcaatgcc
     3961 tccaatgcgc tgaccctggc tcaccagctg cgcgtgtaca cagccgaggc cgctagcttt
     4021 tcagacatga tggatgtagt ctatgtggct cagatgatcc agaaattttt gggttatgtc
     4081 gaccagatca aagagctggt agaggtgatg gtggacatgg ccagcaacct gatgctggtg
     4141 gacgagcacc tgctgtggct ggcccagcgc gaggacaagg cctgcagccg catcgtgggt
     4201 gccctggagc gcattggggg ggccgccctc agcccccatg cccagcacat ctcagtgaat
     4261 gcgaggaacg tggcattgga ggcctacctc atcaagccgc acagctacgt gggcctgacc
     4321 tgcacagcct tccagaggag ggagggaggg gtgccgggca cacggccagg aagccctggc
     4381 cagaaccccc cacctgagcc cgagccccca gctgaccagc agctccgctt ccgctgcacc
     4441 accgggaggc ccaatgtttc tctgtcgtcc ttccacatca agaacagcgt ggccctggcc
     4501 tccatccagc tgcccccgag tctattctca tcccttccgg ctgccctggc tcccccggtg
     4561 cccccagact gcaccctgca actgctcgtc ttccgaaatg gccgcctctt ccacagccac
     4621 agcaacacct cccgccctgg agctgctggg cctggcaaga ggcgtggcgt ggccaccccc
     4681 gtcatcttcg caggaaccag tggctgtggc gtgggaaacc tgacagagcc agtggccgtt
     4741 tcgctgcggc actgggctga gggagccgaa cctgtggccg cttggtggag ccaggagggg
     4801 cccggggagg ctgggggctg gacctcggag ggctgccagc tccgctccag ccagcccaat
     4861 gtcagcgccc tgcactgcca gcacttgggc aatgtggccg tgctcatgga gctggatggg
     4921 gggggttctc atcatcatca tcatcattaa ggatcccggg tggcatccct gtgacccctc
     4981 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     5041 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     5101 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     5161 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     5221 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     5281 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     5341 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     5401 acaggcgtga accactgctc ccttccctgt ccttctgatt ttaaaataac tataccagca
     5461 ggaggacgtc cagacacagc ataggctacc tggccatgcc caaccggtgg gacatttgag
     5521 ttgcttgctt ggcactgtcc tctcatgcgt tgggtccact cagtagatgc ctgttgaatt
     5581 gggtacgcgg ccagcttggc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     5641 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     5701 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     5761 aaccatagtc ccgcccctaa ctccgcccat cccgcctccg ctaactccgc ccagttccgc
     5821 ccattctccg ccccatggct gactaatttt ttttatttat gcaggccgag gccgcctcgg
     5881 cctctgagct attccagaag tagtgaggag gcttttttgg aggaattgat cagcttggga
     5941 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     6001 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     6061 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     6121 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     6181 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     6241 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     6301 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     6361 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     6421 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     6481 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     6541 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     6601 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     6661 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     6721 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     6781 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     6841 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     6901 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc cgggctcgat cccctcgcga
     6961 gttggttcag ctgctgcctg aggctggacg acctcgcgga gttctaccgg cagtgcaaat
     7021 ccgtcggcat ccaggaaacc agcagcggct atccgcgcat ccatgccccc gaactgcagg
     7081 agtggggagg cacgatggcc gctttggtcg acccggacgg gacgctcctg cgcctgatac
     7141 agaacgaatt gcttgcaggc atctcatgag tgtgtcttcc cgttttccgc ctgaggtcac
     7201 tgcgtggatg gagcgctggc gcctgctgcg cgacggcgag ctgctcacca cccactcgcc
     7261 aagctggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gccgatcata
     7321 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     7381 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     7441 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     7501 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcaattcc
     7561 ctatagtgag tcgtattaaa ttcgtaatca tgtcatagct gtttcctg
//
