LOCUS       sequence-496939-        7671 bp    DNA     circular SYN 12-JAN-2026
DEFINITION  Expression of GPR161 V129E mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7671)
  AUTHORS   Krantz S, Bell B, Lund K, Parra NS, Ng Y, De Oliveira Rosa N, 
            Mukhopadhyay S, Croix BS, Sarin KY, Weigert R, Raimondi F, 
            Iglesias-Bartolome R
  TITLE     Dissection of Galphas and Hedgehog signaling crosstalk reveals 
            therapeutic opportunities to target adenosine receptor 2b in 
            Hedgehog-dependent tumors.
  JOURNAL   bioRxiv [Preprint]. 2025 Feb 27:2025.02.21.639530. doi: 
            10.1101/2025.02.21.639530.
  PUBMED    40060632
REFERENCE   2  (bases 1 to 7671)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 12, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Feb 27:2025.02.21.639530. doi: 
            10.1101/2025.02.21.639530."
COMMENT     Sequence Label: pCEFL_GPR161-HA_V129E
FEATURES             Location/Qualifiers
     source          1..7671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..33
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(252..356)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(435..454)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        685..1867
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          916..1858
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1815..1835
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3533..3559
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(3611..3629)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3612..3629)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3649..3666)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3655..3879
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3925..4353
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4012..4031)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4222..4243
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4362..4382)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4367..4696
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4547..4682
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4609..4628
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4763..5557
                     /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(4817..4836)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5427..5446
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5731..5852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5768..5787)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5822..5841
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5901..5917)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5901..5917)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5914..5936)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5925..5941
                     /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(5949..5955,5956..5973,5974..5979))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5994..6015
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6132..6149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6303..6891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6383..6402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7062..7671,1..182,183..251))
                     /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"
ORIGIN
        1 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
       61 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      121 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      181 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
      241 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
      301 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
      361 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta
      421 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
      481 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
      541 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcga
      601 gctttgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc
      661 taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg
      721 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg
      781 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
      841 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
      901 gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
      961 tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc
     1021 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
     1081 cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg
     1141 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
     1201 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatcgatct
     1261 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
     1321 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
     1381 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
     1441 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg
     1501 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     1561 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     1621 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt
     1681 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     1741 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     1801 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     1861 gtcgtgagga attggcgaag ctcagttaac ggccattgtg gccatcaagc ttggtaccag
     1921 atctggatcc gccgccacca tgagcctgaa cagcagcttg agttgtcgca aagagcttag
     1981 taatctgact gaagaagagg gaggtgaagg gggagtaatc attacacagt tcatagctat
     2041 tatagtcata accattttcg tttgcctcgg gaatctcgtc atagtagtta cactgtacaa
     2101 aaaatcttac ttgcttaccc tgtccaacaa gtttgtattt tccctgaccc tctctaactt
     2161 ccttctttct gtgcttgtac tccccttcgt agtaacgtct tcaataagga gggagtggat
     2221 tttcggagta gtatggtgca atttttcagc gctgctgtat ctgcttataa gtagcgcaag
     2281 tatgcttacg ctcggggtta tcgccattga tcgatattac gcggaattgt acccaatggt
     2341 ataccctatg aaaataacgg gaaaccgagc cgtcatggca ctcgtttaca tttggctcca
     2401 ctcactcatt ggctgtctcc cgccgctgtt cgggtggagc agtgtcgagt tcgacgaatt
     2461 taagtggatg tgcgtcgcgg cttggcaccg ggagccaggc tatacggcct tttggcaaat
     2521 ttggtgcgct ttgttcccgt ttttggtaat gctggtgtgt tatggcttta tcttccgcgt
     2581 ggcgcgagtc aaagcccgaa aggttcattg cggaacggtt gtcattgtag aggaagatgc
     2641 tcaacgcacg ggccggaaaa actcttctac cagcacgtca tcttctggca gtcgacggaa
     2701 tgcattccaa ggggtagtgt actccgccaa ccaatgcaag gctctgatca cgattctggt
     2761 ggtacttggc gcgttcatgg tgacctgggg gccgtatatg gtcgttattg cgtccgaggc
     2821 tctctgggga aagtcttcag tatctcccag cctggaaact tgggcaacgt ggctttcttt
     2881 cgcgtccgct gtctgccatc cattgattta tggattgtgg aataaaaccg tccgaaagga
     2941 attgcttggt atgtgcttcg gtgatcgcta ttacagagaa ccctttgtgc agcgacagcg
     3001 gaccagccga ttgttttcta taagcaatcg gattacagac cttgggctgt ccccacacct
     3061 taccgcgctt atggctggag ggcaaccctt ggggcattca agttctactg gtgacacggg
     3121 cttttcatgt tcccaggatt caggtactga tatgatgctt ctggaagact acacgtccga
     3181 cgataatccc cccagtcatt gtacatgtcc gcccaaacgc cgatcctccg tgacttttga
     3241 ggatgaagtg gagcaaataa aagaagctgc taagaacagc attctgcatg ttaaagctga
     3301 ggtccacaag tcactcgata gctatgcggc tagcctcgcc aaggcgatcg aagcagaggc
     3361 caagatcaac ctgttcggcg aagaggcgct cccaggcgtc ctggtaacag ctcggaccgt
     3421 tccaggtggg ggcttcggtg gtagacgcgg cagcagaact ctggtctccc agaggctgca
     3481 gctgcaaagc attgaagaag gcgatgtgct cgcagccgaa caacgggaat tctacccata
     3541 cgatgttcct gactatgcgt aggcggccgc actcgagata tcggcctcca gggcctctag
     3601 agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc
     3661 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3721 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3781 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3841 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     3901 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3961 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4021 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4081 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4141 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4201 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     4261 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     4321 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     4381 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4441 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4501 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     4561 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4621 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4681 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     4741 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4801 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4861 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4921 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4981 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     5041 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     5101 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5161 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5221 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5281 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5341 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5401 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5461 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5521 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5581 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5641 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5701 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     5761 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5821 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5881 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5941 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     6001 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6061 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6121 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6181 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6241 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6301 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6361 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6421 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6481 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6541 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6601 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6661 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6721 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6781 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6841 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6901 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6961 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7021 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7081 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7141 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7201 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7261 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7321 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7381 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7441 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7501 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7561 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7621 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc g
//
