LOCUS       sequence-497381-        7242 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Expression of Galphas T350I mutant (GNAST350I) 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 T350I
FEATURES             Location/Qualifiers
     source          1..7242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..32
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(251..355)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(434..453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        684..1866
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          915..1857
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1814..1834
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1939..1948
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3181..3199)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3182..3199)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3219..3236)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3225..3449
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3495..3923
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3582..3601)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3792..3813
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3932..3952)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3937..4266
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4117..4252
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4179..4198
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4333..5127
                     /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(4387..4406)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4997..5016
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5301..5422
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5338..5357)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5392..5411
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5471..5487)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5471..5487)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5484..5506)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5495..5511
                     /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(5519..5525,5526..5543,5544..5549))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5564..5585
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5702..5719)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5873..6461)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5953..5972)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6632..7242,1..181,182..250))
                     /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 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
       61 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
      121 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
      181 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
      241 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
      301 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
      361 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctat
      421 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
      481 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
      541 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgag
      601 ctttgcaaag atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct
      661 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc
      721 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
      781 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
      841 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
      901 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt
      961 atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg
     1021 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc
     1081 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc
     1141 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac
     1201 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatcgatctg
     1261 cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc
     1321 acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct
     1381 caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg
     1441 gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc
     1501 cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca
     1561 cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag
     1621 taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta
     1681 ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa
     1741 gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga
     1801 tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg
     1861 tcgtgaggaa ttggcgaagc tcagttaacg gccattgtgg ccatcaagct tggtaccaga
     1921 tctggatccg ctagcgccgc caccatgggc tgcctcggga acagtaagac cgaggaccag
     1981 cgcaacgagg agaaggcgca gcgtgaggcc aacaaaaaga tcgagaagca gctgcagaag
     2041 gacaagcagg tctaccgggc cacgcaccgc ctgctccttt tgggcgctgg agaatctggt
     2101 aaaagcacca ttgtgaagca gatgaggatc ctgcatgtta atgggtttaa tggagagggc
     2161 ggcgaagagg acccgcaggc tgcaaggagc aacagcgatg gtgagaaggc aaccaaagtg
     2221 caggacatca aaaacaacct gaaagaggcg attgaaacca ttgtggccgc catgagcaac
     2281 ctggtgcccc ccgtggagct ggccaacccc gagaaccagt tcagagtgga ctacatcctg
     2341 agtgtgatga acgtgcctga ctttgacttc cctcccgaat tctatgagca tgccaaggct
     2401 ctgtgggagg atgaaggagt gcgtgcctgc tacgaacgct ccaacgagta ccagctgatt
     2461 gactgtgccc agtacttcct ggacaagatc gacgtgatca agcaggctga atatatgccg
     2521 accgagcagg acctgcttcg ctgccgtgtc ctgacttctg gaatctttga gaccaagttc
     2581 caggtggaca aagtcaactt ccacatgttt gacgtgggtg gccagcgcga tgaacgccgc
     2641 aagtggatcc agtgcttcaa cgatgtgact gccatcatct tcgtggtggc cagcagcagc
     2701 tacaacatgg tcatccggga ggacaaccag accaaccgcc tgcaggaggc tctgaacctc
     2761 ttcaagagca tctggaacaa cagatggctg cgcaccatct ctgtgatcct gttcctcaac
     2821 aagcaagatc tgctcgctga gaaagtcctt gctgggaaat cgaagattga ggactacttt
     2881 ccagaatttg ctcgctacac tactcctgag gatgctactc ccgagcccgg agaggaccca
     2941 cgcgtgaccc gggccaagta cttcattcga gatgagtttc tgaggatcag cattgccagt
     3001 ggagatgggc gtcactactg ctaccctcat ttcacctgcg ctgtggacac tgagaacatc
     3061 cgccgtgtgt tcaacgactg ccgtgacatc attcagcgca tgcaccttcg tcagtacgag
     3121 ctgctctaat aggcggccgc actcgagata tcggcctcca gggcctctag agggccctat
     3181 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     3241 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3301 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3361 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3421 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3481 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3541 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3601 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3661 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3721 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3781 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3841 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3901 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3961 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4021 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4081 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4141 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4201 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4261 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     4321 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4381 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4441 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4501 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4561 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4621 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4681 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4741 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4801 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4861 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4921 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4981 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5041 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     5101 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5161 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5221 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5281 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     5341 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5401 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5461 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5521 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5581 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5641 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5701 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5761 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5821 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5881 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5941 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6001 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6061 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6121 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6181 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6241 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6301 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6361 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6421 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6481 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6541 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6601 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6661 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6721 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6781 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6841 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6901 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6961 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7021 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7081 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7141 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7201 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc gg
//
