LOCUS       sequence-497387-        7137 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Expression of PKA R135W mutant (PRKACAR135W).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7137)
  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 7137)
  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 PRKACA HA R135W
FEATURES             Location/Qualifiers
     source          1..7137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(105..209)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(288..307)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        538..1720
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          769..1711
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1668..1688
                     /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"
     CDS             2852..2878
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(2930..2948)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2931..2948)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2968..2985)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2974..3198
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3244..3672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3331..3350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3541..3562
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3681..3701)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3686..4015
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3866..4001
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3928..3947
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4082..4876
                     /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(4136..4155)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4746..4765
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5050..5171
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5087..5106)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5141..5160
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5220..5236)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5220..5236)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5233..5255)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5244..5260
                     /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(5268..5274,5275..5292,5293..5298))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5313..5334
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5451..5468)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5622..6210)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5702..5721)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6381..7137,1..35,36..104))
                     /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     7004..7023
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
       61 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
      121 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
      181 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
      241 gacggatcgg gagatcgatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
      301 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      361 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      421 ctgcttaggg ttaggcgttt tgcgctgctt cgagctttgc aaagatggat aaagttttaa
      481 acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt gggaattggc
      541 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
      601 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
      661 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
      721 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg
      781 tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact
      841 tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag
      901 agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct
      961 gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc
     1021 gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa
     1081 gatagtcttg taaatgcggg ccaagatcga tctgcacact ggtatttcgg tttttggggc
     1141 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
     1201 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
     1261 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
     1321 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
     1381 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     1441 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     1501 ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     1561 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     1621 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     1681 agtggttcaa agtttttttc ttccatttca ggtgtcgtga ggaattggcg aagctcagtt
     1741 aacggccatt gtggccatca agcttggtac cagatctgga tccgccgcca ccatgggcaa
     1801 cgccgccgcc gccaagaagg gcagcgagca ggagagcgtg aaagaattct tagccaaagc
     1861 caaagaagat tttcttaaaa aatgggaaag tcccgctcag aacacagccc acttggatca
     1921 gtttgaacga atcaagaccc tcggcacggg ctccttcggg cgggtgatgc tggtgaaaca
     1981 caaggagacc gggaaccact atgccatgaa gatcctcgac aaacagaagg tggtgaaact
     2041 gaaacagatc gaacacaccc tgaatgaaaa gcgcatcctg caagctgtca actttccgtt
     2101 cctcgtcaaa ctcgagttct ccttcaagga caactcaaac ttatacatgg tcatggagta
     2161 cgtgcccggc ggggagatgt tctcacacct acggtggatc ggaaggttca gtgagcccca
     2221 tgcccgtttc tacgcggccc agatcgtcct gacctttgag tatctgcact cgctggatct
     2281 catctacagg gacctgaagc cggagaatct gctcattgac cagcagggct acattcaggt
     2341 gacagacttc ggtttcgcca agcgcgtgaa gggccgcact tggaccttgt gcggcacccc
     2401 tgagtacctg gcccctgaga ttatcctgag caaaggctac aacaaggccg tggactggtg
     2461 ggccctgggg gttcttatct atgaaatggc cgctggctac ccgcccttct tcgcagacca
     2521 gcccatccag atctatgaga agatcgtctc tgggaaggtg cgcttccctt cccacttcag
     2581 ctctgacttg aaggacctgc tgcggaacct cctgcaggta gatctcacca agcgctttgg
     2641 gaacctcaag aatggggtca acgatatcaa gaaccacaag tggtttgcca caactgactg
     2701 gattgccatc taccagagga aggtggaagc tcccttcata ccaaagttta aaggccctgg
     2761 ggatacgagt aactttgacg actatgagga agaagaaatc cgggtctcca tcaatgagaa
     2821 gtgtggcaag gagttttctg agtttgctag ctacccatac gacgtgccag attacgccta
     2881 ggcggccgca ctcgagatat cggcctccag ggcctctaga gggccctatt ctatagtgtc
     2941 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3001 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3061 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3121 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3181 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3241 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3301 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3361 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3421 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3481 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3541 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3601 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3661 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3721 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3781 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3841 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3901 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3961 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4021 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     4081 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4141 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4201 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4261 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4321 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4381 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4441 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4501 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4561 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4621 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4681 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4741 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4801 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4861 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4921 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4981 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5041 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5101 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5161 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5221 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5281 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5341 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5401 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5461 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5521 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5581 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5641 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5701 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5761 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5821 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5881 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5941 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6001 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6061 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6121 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6181 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6241 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6301 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6361 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6421 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6481 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6541 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6601 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6661 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6721 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6781 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6841 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6901 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6961 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7021 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7081 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatct
//
