LOCUS       sequence-496937-        7242 bp    DNA     circular SYN 12-JAN-2026
DEFINITION  Expression of Galphas K32R mutant (GNASK32R) EE-tagged (internal).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7242)
  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 7242)
  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 GNAS EE K32R
FEATURES             Location/Qualifiers
     source          1..7242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          143..1085
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1042..1062
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1167..1176
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2409..2427)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2410..2427)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2447..2464)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2453..2677
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2723..3151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2810..2829)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3020..3041
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3160..3180)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3165..3494
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3345..3480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3407..3426
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3561..4355
                     /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(3615..3634)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4225..4244
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4529..4650
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4566..4585)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4620..4639
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4699..4715)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4699..4715)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4712..4734)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4723..4739
                     /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(4747..4753,4754..4771,4772..4777))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4792..4813
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4930..4947)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5101..5689)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5181..5200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5860..6651,6652..6720))
                     /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     6483..6502
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6721..6825)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6904..6923)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7154..7242,1..1094)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
ORIGIN
        1 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
       61 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
      121 cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt
      181 tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct
      241 tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc
      301 cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat
      361 ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt
      421 ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga
      481 tcgatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt
      541 cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg
      601 ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc
      661 cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc
      721 ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg
      781 gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact
      841 ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt
      901 cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg
      961 agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg
     1021 agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat
     1081 ttcaggtgtc gtgaggaatt ggcgaagctc agttaacggc cattgtggcc atcaagcttg
     1141 gtaccagatc tggatccgct agcgccgcca ccatgggctg cctcgggaac agtaagaccg
     1201 aggaccagcg caacgaggag aaggcgcagc gtgaggccaa caaaaagatc gagaagcagc
     1261 tgcagaggga caagcaggtc taccgggcca cgcaccgcct gctccttttg ggcgctggag
     1321 aatctggtaa aagcaccatt gtgaagcaga tgaggatcct gcatgttaat gggtttaatg
     1381 gagagggcgg cgaagaggac ccgcaggctg caaggagcaa cagcgatggt gagaaggcaa
     1441 ccaaagtgca ggacatcaaa aacaacctga aagaggcgat tgaaaccatt gtggccgcca
     1501 tgagcaacct ggtgcccccc gtggagctgg ccaaccccga gaaccagttc agagtggact
     1561 acatcctgag tgtgatgaac gtgcctgact ttgacttccc tcccgaattc tatgagcatg
     1621 ccaaggctct gtgggaggat gaaggagtgc gtgcctgcta cgaacgctcc aacgagtacc
     1681 agctgattga ctgtgcccag tacttcctgg acaagatcga cgtgatcaag caggctgaat
     1741 atatgccgac cgagcaggac ctgcttcgct gccgtgtcct gacttctgga atctttgaga
     1801 ccaagttcca ggtggacaaa gtcaacttcc acatgtttga cgtgggtggc cagcgcgatg
     1861 aacgccgcaa gtggatccag tgcttcaacg atgtgactgc catcatcttc gtggtggcca
     1921 gcagcagcta caacatggtc atccgggagg acaaccagac caaccgcctg caggaggctc
     1981 tgaacctctt caagagcatc tggaacaaca gatggctgcg caccatctct gtgatcctgt
     2041 tcctcaacaa gcaagatctg ctcgctgaga aagtccttgc tgggaaatcg aagattgagg
     2101 actactttcc agaatttgct cgctacacta ctcctgagga tgctactccc gagcccggag
     2161 aggacccacg cgtgacccgg gccaagtact tcattcgaga tgagtttctg aggatcagca
     2221 ctgccagtgg agatgggcgt cactactgct accctcattt cacctgcgct gtggacactg
     2281 agaacatccg ccgtgtgttc aacgactgcc gtgacatcat tcagcgcatg caccttcgtc
     2341 agtacgagct gctctaatag gcggccgcac tcgagatatc ggcctccagg gcctctagag
     2401 ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct
     2461 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2521 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2581 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     2641 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     2701 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2761 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2821 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2881 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2941 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3001 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3061 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3121 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3181 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3241 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3301 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3361 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3421 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3481 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     3541 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3601 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3661 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3721 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3781 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3841 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3901 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3961 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4021 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4081 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4141 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4201 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4261 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4321 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     4381 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     4441 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4501 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4561 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4621 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4681 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4741 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4801 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4861 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4921 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4981 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5041 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5101 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5161 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5221 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5281 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5341 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5401 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5461 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5521 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5581 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5641 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5701 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5761 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5821 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5881 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5941 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6001 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6061 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6121 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6181 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6241 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6301 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6361 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6421 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6481 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6541 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6601 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6661 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6721 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6781 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6841 agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctat ggtgcactct
     6901 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     6961 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     7021 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgag ctttgcaaag
     7081 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa
     7141 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     7201 cgagaagttg gggggagggg tcggcaattg aaccggtgcc ta
//
