LOCUS       sequence-497390-        7137 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Expression of PKA E171K mutant (PRKACAE171K).
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 E171K
FEATURES             Location/Qualifiers
     source          1..7137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(48..67)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        298..1480
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          529..1471
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1428..1448
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1547..1556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2612..2638
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(2690..2708)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2691..2708)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2728..2745)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2734..2958
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3004..3432
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3091..3110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3301..3322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3441..3461)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3446..3775
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3626..3761
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3688..3707
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3842..4636
                     /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(3896..3915)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4506..4525
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4810..4931
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4847..4866)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4901..4920
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4980..4996)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4980..4996)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4993..5015)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5004..5020
                     /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(5028..5034,5035..5052,5053..5058))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5073..5094
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5211..5228)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5382..5970)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5462..5481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6141..6932,6933..7001))
                     /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     6764..6783
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7002..7106)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcgatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
       61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      181 ctgcttaggg ttaggcgttt tgcgctgctt cgagctttgc aaagatggat aaagttttaa
      241 acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt gggaattggc
      301 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
      361 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
      421 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
      481 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg
      541 tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact
      601 tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag
      661 agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct
      721 gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc
      781 gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa
      841 gatagtcttg taaatgcggg ccaagatcga tctgcacact ggtatttcgg tttttggggc
      901 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
      961 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
     1021 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
     1081 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
     1141 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     1201 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     1261 ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     1321 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     1381 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     1441 agtggttcaa agtttttttc ttccatttca ggtgtcgtga ggaattggcg aagctcagtt
     1501 aacggccatt gtggccatca agcttggtac cagatctgga tccgccgcca ccatgggcaa
     1561 cgccgccgcc gccaagaagg gcagcgagca ggagagcgtg aaagaattct tagccaaagc
     1621 caaagaagat tttcttaaaa aatgggaaag tcccgctcag aacacagccc acttggatca
     1681 gtttgaacga atcaagaccc tcggcacggg ctccttcggg cgggtgatgc tggtgaaaca
     1741 caaggagacc gggaaccact atgccatgaa gatcctcgac aaacagaagg tggtgaaact
     1801 gaaacagatc gaacacaccc tgaatgaaaa gcgcatcctg caagctgtca actttccgtt
     1861 cctcgtcaaa ctcgagttct ccttcaagga caactcaaac ttatacatgg tcatggagta
     1921 cgtgcccggc ggggagatgt tctcacacct acggcggatc ggaaggttca gtgagcccca
     1981 tgcccgtttc tacgcggccc agatcgtcct gacctttgag tatctgcact cgctggatct
     2041 catctacagg gacctgaagc cgaagaatct gctcattgac cagcagggct acattcaggt
     2101 gacagacttc ggtttcgcca agcgcgtgaa gggccgcact tggaccttgt gcggcacccc
     2161 tgagtacctg gcccctgaga ttatcctgag caaaggctac aacaaggccg tggactggtg
     2221 ggccctgggg gttcttatct atgaaatggc cgctggctac ccgcccttct tcgcagacca
     2281 gcccatccag atctatgaga agatcgtctc tgggaaggtg cgcttccctt cccacttcag
     2341 ctctgacttg aaggacctgc tgcggaacct cctgcaggta gatctcacca agcgctttgg
     2401 gaacctcaag aatggggtca acgatatcaa gaaccacaag tggtttgcca caactgactg
     2461 gattgccatc taccagagga aggtggaagc tcccttcata ccaaagttta aaggccctgg
     2521 ggatacgagt aactttgacg actatgagga agaagaaatc cgggtctcca tcaatgagaa
     2581 gtgtggcaag gagttttctg agtttgctag ctacccatac gacgtgccag attacgccta
     2641 ggcggccgca ctcgagatat cggcctccag ggcctctaga gggccctatt ctatagtgtc
     2701 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2761 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2821 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2881 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2941 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3001 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3061 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3421 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3481 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3541 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3601 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3661 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3721 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3781 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3841 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3901 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3961 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4021 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4081 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4141 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4201 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4261 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4321 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4381 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4441 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4501 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4561 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4621 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4681 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4741 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4801 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4861 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4921 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4981 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5041 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5101 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5161 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5221 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5281 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5341 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5401 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5461 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5521 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5581 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5641 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5701 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5761 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5821 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5881 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5941 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6001 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6061 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6121 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6181 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6241 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6301 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6361 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6421 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6481 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6541 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6601 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6661 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6721 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6781 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6841 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6901 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6961 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7021 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7081 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
