LOCUS       sequence-497393-        7242 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Expression of Galphas P116L mutant (GNASP116L) 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 P116L
FEATURES             Location/Qualifiers
     source          1..7242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(106..125)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      919..1507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1408..1427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1661..1678
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1795..1816
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1831..1836,1837..1854,1855..1861)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1869..1885
                     /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)."
     primer_bind     1874..1896
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1893..1909
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1893..1909
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1958..2079
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1969..1988)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2023..2042
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2253..3047)
                     /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(2364..2383)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2974..2993
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3114..3443)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3128..3263
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3182..3201)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3428..3448
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3457..3885)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3567..3588)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3779..3798
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3931..4155
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4144..4161
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        4181..4199
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4181..4198
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      5432..5441
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5514..6696)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5523..6465
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(5546..5566)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     6927..6946
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7025..7129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7130..7198,7199..7242,1..748)
                     /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 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
       61 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
      121 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
      181 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
      241 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
      301 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
      361 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
      421 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
      481 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
      541 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
      601 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
      661 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
      721 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
      781 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
      841 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
      901 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
      961 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1021 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     1081 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1141 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1201 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1261 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1321 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1381 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1441 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1501 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     1561 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     1621 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     1681 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     1741 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     1801 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     1861 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     1921 caagctctag ctagaggtcg acggtataca gacatgataa gatacattga tgagtttgga
     1981 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     2041 gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga agaactccag
     2101 catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc
     2161 caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc
     2221 ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag
     2281 aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc
     2341 cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg
     2401 tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg
     2461 atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgcgc
     2521 gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca
     2581 tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct
     2641 tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc
     2701 atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact
     2761 tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa
     2821 ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg
     2881 gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac
     2941 acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc
     3001 acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct
     3061 catcctgtct cttgatcaga tccgaaaatg gatatacaag ctcccgggag ctttttgcaa
     3121 aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggcagaggcg
     3181 gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa
     3241 ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa
     3301 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg
     3361 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     3421 tttccacacc ctaactgaca cacattccac agaattaatt cgcgttaaat ttttgttaaa
     3481 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3541 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3601 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3661 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3721 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3781 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3841 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtgggga taccccctag
     3901 agccccagct ggttctttcc gcctcagaag ccatagagcc caccgcatcc ccagcatgcc
     3961 tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc caccccaccc cccagaatag
     4021 aatgacacct actcagacaa tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg
     4081 gagtggcacc ttccagggtc aaggaaggca cgggggaggg gcaaacaaca gatggctggc
     4141 aactagaagg cacagtcgag gctgatcagc gagctctagc atttaggtga cactatagaa
     4201 tagggccctc tagaggccct ggaggccgat atctcgagtg cggccgccta ttagagcagc
     4261 tcgtactgac gaaggtgcat gcgctgaatg atgtcacggc agtcgttgaa cacacggcgg
     4321 atgttctcag tgtccacagc gcaggtgaaa tgagggtagc agtagtgacg cccatctcca
     4381 ctggcagtgc tgatcctcag aaactcatct cgaatgaagt acttggcccg ggtcacgcgt
     4441 gggtcctctc cgggctcggg agtagcatcc tcaggagtag tgtagcgagc aaattctgga
     4501 aagtagtcct caatcttcga tttcccagca aggactttct cagcgagcag atcttgcttg
     4561 ttgaggaaca ggatcacaga gatggtgcgc agccatctgt tgttccagat gctcttgaag
     4621 aggttcagag cctcctgcag gcggttggtc tggttgtcct cccggatgac catgttgtag
     4681 ctgctgctgg ccaccacgaa gatgatggca gtcacatcgt tgaagcactg gatccacttg
     4741 cggcgttcat cgcgctggcc acccacgtca aacatgtgga agttgacttt gtccacctgg
     4801 aacttggtct caaagattcc agaagtcagg acacggcagc gaagcaggtc ctgctcggtc
     4861 ggcatatatt cagcctgctt gatcacgtcg atcttgtcca ggaagtactg ggcacagtca
     4921 atcagctggt actcgttgga gcgttcgtag caggcacgca ctccttcatc ctcccacaga
     4981 gccttggcat gctcatagaa ttcgggaggg aagtcaaagt caggcacgtt catcacactc
     5041 aggatgtagt ccactctgaa ctggttctcg gggttggcca gctccacgag gggcaccagg
     5101 ttgctcatgg cggccacaat ggtttcaatc gcctctttca ggttgttttt gatgtcctgc
     5161 actttggttg ccttctcacc atcgctgttg ctccttgcag cctgcgggtc ctcttcgccg
     5221 ccctctccat taaacccatt aacatgcagg atcctcatct gcttcacaat ggtgctttta
     5281 ccagattctc cagcgcccaa aaggagcagg cggtgcgtgg cccggtagac ctgcttgtcc
     5341 ttctgcagct gcttctcgat ctttttgttg gcctcacgct gcgccttctc ctcgttgcgc
     5401 tggtcctcgg tcttactgtt cccgaggcag cccatggtgg cggcgctagc ggatccagat
     5461 ctggtaccaa gcttgatggc cacaatggcc gttaactgag cttcgccaat tcctcacgac
     5521 acctgaaatg gaagaaaaaa actttgaacc actgtctgag gcttgagaat gaaccaagat
     5581 ccaaactcaa aaagggcaaa ttccaaggag aattacatca agtgccaagc tggcctaact
     5641 tcagtctcca cccactcagt gtggggaaac tccatcgcat aaaacccctc cccccaacct
     5701 aaagacgacg tactccaaaa gctcgagaac taatcgaggt gcctggacgg cgcccggtac
     5761 tccgtggagt cacatgaagc gacggctgag gacggaaagg cccttttcct ttgtgtgggt
     5821 gactcacccg cccgctctcc cgagcgccgc gtcctccatt ttgagctccc tgcagcaggg
     5881 ccgggaagcg gccatctttc cgctcacgca actggtgccg accgggccag ccttgccgcc
     5941 cagggcgggg cgatacacgg cggcgcgagg ccaggcacca gagcaggccg gccagcttga
     6001 gactaccccc gtccgattct cggtggccgc gctcgcaggc cccgcctcgc cgaacatgtg
     6061 cgctgggacg cacgggcccc gtcgccgccc gcggccccaa aaaccgaaat accagtgtgc
     6121 agatcgatct tggcccgcat ttacaagact atcttgccag aaaaaaagcg tcgcagcagg
     6181 tcatcaaaaa ttttaaatgg ctagagactt atcgaaagca gcgagacagg cgcgaaggtg
     6241 ccaccagatt cgcacgcggc ggccccagcg cccaggccag gcctcaactc aagcacgagg
     6301 cgaaggggct ccttaagcgc aaggcctcga actctcccac ccacttccaa cccgaagctc
     6361 gggatcaaga atcacgtact gcagccaggt ggaagtaatt caaggcacgc aagggccata
     6421 acccgtaaag aggccaggcc cgcgggaacc acacacggca cttacctgtg ttctggcggc
     6481 aaacccgttg cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc
     6541 accctcggga aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca
     6601 ccttctctag gcaccggttc aattgccgac ccctcccccc aacttctcgg ggactgtggg
     6661 cgatgtgcgc tctgcccact gacgggcacc ggagccaatt cccactcctt tcaagaccta
     6721 gaaggtccat tagctgcaaa gattcctctc tgtttaaaac tttatccatc tttgcaaagc
     6781 tcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaattgt cggtcaagcc
     6841 ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc
     6901 agggagcaga tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     6961 taggggatcg ggagatcgat ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa
     7021 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     7081 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     7141 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     7201 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag tt
//
