LOCUS       sequence-497397-        7242 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Expression of Galphas N218I mutant (GNASN218I) EE-tagged (internal).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7242)
  AUTHORS   Krantz S, Bell BA, Lund K, Salinas Parra N, Ng Y, De Oliveira Rosa 
            N, Mukhopadhyay S, St Croix B, Sarin KY, Weigert R, Raimondi F, 
            Iglesias-Bartolome R
  TITLE     Dissection of Galphas and Hedgehog Signaling Crosstalk Reveals 
            Therapeutic Opportunities to Target Hedgehog-Dependent Tumors.
  JOURNAL   Cancer Res. 2025 Dec 29. doi: 10.1158/0008-5472.CAN-25-1109.
  PUBMED    41460725
REFERENCE   2  (bases 1 to 7242)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 15, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cancer Res.
            2025 Dec 29. doi: 10.1158/0008-5472.CAN-25-1109."
COMMENT     Sequence Label: pCEFL GNAS EE N218I
FEATURES             Location/Qualifiers
     source          1..7242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(99..203)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(282..301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        532..1714
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          763..1705
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1662..1682
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1787..1796
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3029..3047)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3030..3047)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3067..3084)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3073..3297
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3343..3771
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3430..3449)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3640..3661
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3780..3800)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3785..4114
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3965..4100
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4027..4046
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4181..4975
                     /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(4235..4254)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4845..4864
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5149..5270
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5186..5205)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5240..5259
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5319..5335)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5319..5335)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5332..5354)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5343..5359
                     /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(5367..5373,5374..5391,5392..5397))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5412..5433
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5550..5567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5721..6309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5801..5820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6480..7242,1..29,30..98))
                     /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     7103..7122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
       61 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
      121 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
      181 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
      241 tcgggagatc gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
      301 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
      361 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
      421 agggttaggc gttttgcgct gcttcgagct ttgcaaagat ggataaagtt ttaaacagag
      481 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
      541 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      601 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      661 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      721 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
      781 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
      841 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
      901 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
      961 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     1021 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     1081 cttgtaaatg cgggccaaga tcgatctgca cactggtatt tcggtttttg gggccgcggg
     1141 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg
     1201 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct
     1261 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc
     1321 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg
     1381 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc
     1441 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt
     1501 ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt
     1561 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc
     1621 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt
     1681 tcaaagtttt tttcttccat ttcaggtgtc gtgaggaatt ggcgaagctc agttaacggc
     1741 cattgtggcc atcaagcttg gtaccagatc tggatccgct agcgccgcca ccatgggctg
     1801 cctcgggaac agtaagaccg aggaccagcg caacgaggag aaggcgcagc gtgaggccaa
     1861 caaaaagatc gagaagcagc tgcagaagga caagcaggtc taccgggcca cgcaccgcct
     1921 gctccttttg ggcgctggag aatctggtaa aagcaccatt gtgaagcaga tgaggatcct
     1981 gcatgttaat gggtttaatg gagagggcgg cgaagaggac ccgcaggctg caaggagcaa
     2041 cagcgatggt gagaaggcaa ccaaagtgca ggacatcaaa aacaacctga aagaggcgat
     2101 tgaaaccatt gtggccgcca tgagcaacct ggtgcccccc gtggagctgg ccaaccccga
     2161 gaaccagttc agagtggact acatcctgag tgtgatgaac gtgcctgact ttgacttccc
     2221 tcccgaattc tatgagcatg ccaaggctct gtgggaggat gaaggagtgc gtgcctgcta
     2281 cgaacgctcc aacgagtacc agctgattga ctgtgcccag tacttcctgg acaagatcga
     2341 cgtgatcaag caggctgaat atatgccgac cgagcaggac ctgcttcgct gccgtgtcct
     2401 gacttctgga atctttgaga ccaagttcca ggtggacaaa gtcatcttcc acatgtttga
     2461 cgtgggtggc cagcgcgatg aacgccgcaa gtggatccag tgcttcaacg atgtgactgc
     2521 catcatcttc gtggtggcca gcagcagcta caacatggtc atccgggagg acaaccagac
     2581 caaccgcctg caggaggctc tgaacctctt caagagcatc tggaacaaca gatggctgcg
     2641 caccatctct gtgatcctgt tcctcaacaa gcaagatctg ctcgctgaga aagtccttgc
     2701 tgggaaatcg aagattgagg actactttcc agaatttgct cgctacacta ctcctgagga
     2761 tgctactccc gagcccggag aggacccacg cgtgacccgg gccaagtact tcattcgaga
     2821 tgagtttctg aggatcagca ctgccagtgg agatgggcgt cactactgct accctcattt
     2881 cacctgcgct gtggacactg agaacatccg ccgtgtgttc aacgactgcc gtgacatcat
     2941 tcagcgcatg caccttcgtc agtacgagct gctctaatag gcggccgcac tcgagatatc
     3001 ggcctccagg gcctctagag ggccctattc tatagtgtca cctaaatgct agagctcgct
     3061 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3121 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3181 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3241 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     3301 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     3361 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3421 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3481 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3541 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3601 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3661 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3721 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     3781 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3841 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3901 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3961 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4021 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     4081 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     4141 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     4201 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     4261 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     4321 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     4381 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     4441 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     4501 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     4561 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     4621 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     4681 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     4741 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     4801 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4861 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4921 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     4981 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     5041 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5101 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     5161 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5221 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     5281 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     5341 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5401 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5461 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5521 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5581 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5641 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5701 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5761 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5821 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5881 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5941 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6001 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6061 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6121 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6181 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6241 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6301 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6361 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6421 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6481 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6541 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6601 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6661 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6721 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6781 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6841 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6901 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6961 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7021 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7081 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7141 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7201 agttcgatgt aacccactcg tgcacccaac tgatcttcag ca
//
