LOCUS       sequence-497386-        7137 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Expression of PKA T49I mutant (PRKACAT49I).
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 T49I
FEATURES             Location/Qualifiers
     source          1..7137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(249..353)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(432..451)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        682..1864
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          913..1855
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1812..1832
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1931..1940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2996..3022
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(3074..3092)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3075..3092)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3112..3129)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3118..3342
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3388..3816
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3475..3494)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3685..3706
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3825..3845)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3830..4159
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4010..4145
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4072..4091
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4226..5020
                     /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(4280..4299)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4890..4909
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5194..5315
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5231..5250)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5285..5304
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5364..5380)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5364..5380)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5377..5399)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5388..5404
                     /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(5412..5418,5419..5436,5437..5442))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5457..5478
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5595..5612)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5766..6354)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5846..5865)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6525..7137,1..179,180..248))
                     /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 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
       61 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      121 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      181 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      241 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      301 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      361 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctatgg
      421 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
      481 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
      541 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgagct
      601 ttgcaaagat ggataaagtt ttaaacagag aggaatcttt gcagctaatg gaccttctag
      661 gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc
      721 acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg
      781 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg
      841 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
      901 gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat
      961 ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag
     1021 cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct
     1081 cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac
     1141 cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct
     1201 gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga tcgatctgca
     1261 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
     1321 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
     1381 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
     1441 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
     1501 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
     1561 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     1621 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     1681 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     1741 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     1801 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     1861 gtgaggaatt ggcgaagctc agttaacggc cattgtggcc atcaagcttg gtaccagatc
     1921 tggatccgcc gccaccatgg gcaacgccgc cgccgccaag aagggcagcg agcaggagag
     1981 cgtgaaagaa ttcttagcca aagccaaaga agattttctt aaaaaatggg aaagtcccgc
     2041 tcagaacaca gcccacttgg atcagtttga acgaatcaag atcctcggca cgggctcctt
     2101 cgggcgggtg atgctggtga aacacaagga gaccgggaac cactatgcca tgaagatcct
     2161 cgacaaacag aaggtggtga aactgaaaca gatcgaacac accctgaatg aaaagcgcat
     2221 cctgcaagct gtcaactttc cgttcctcgt caaactcgag ttctccttca aggacaactc
     2281 aaacttatac atggtcatgg agtacgtgcc cggcggggag atgttctcac acctacggcg
     2341 gatcggaagg ttcagtgagc cccatgcccg tttctacgcg gcccagatcg tcctgacctt
     2401 tgagtatctg cactcgctgg atctcatcta cagggacctg aagccggaga atctgctcat
     2461 tgaccagcag ggctacattc aggtgacaga cttcggtttc gccaagcgcg tgaagggccg
     2521 cacttggacc ttgtgcggca cccctgagta cctggcccct gagattatcc tgagcaaagg
     2581 ctacaacaag gccgtggact ggtgggccct gggggttctt atctatgaaa tggccgctgg
     2641 ctacccgccc ttcttcgcag accagcccat ccagatctat gagaagatcg tctctgggaa
     2701 ggtgcgcttc ccttcccact tcagctctga cttgaaggac ctgctgcgga acctcctgca
     2761 ggtagatctc accaagcgct ttgggaacct caagaatggg gtcaacgata tcaagaacca
     2821 caagtggttt gccacaactg actggattgc catctaccag aggaaggtgg aagctccctt
     2881 cataccaaag tttaaaggcc ctggggatac gagtaacttt gacgactatg aggaagaaga
     2941 aatccgggtc tccatcaatg agaagtgtgg caaggagttt tctgagtttg ctagctaccc
     3001 atacgacgtg ccagattacg cctaggcggc cgcactcgag atatcggcct ccagggcctc
     3061 tagagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg
     3121 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3181 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3241 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3301 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3361 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3421 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3481 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3541 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3601 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3661 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3721 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3781 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3841 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3901 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3961 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4021 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4081 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     4141 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4201 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4261 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4321 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4381 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4441 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4501 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4561 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4621 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4681 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4741 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4801 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4861 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4921 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4981 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     5041 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     5101 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5161 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     5221 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5281 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5341 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5401 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5461 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5521 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5581 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5641 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5701 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5761 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5821 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5881 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5941 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6001 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6061 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6121 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6181 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6241 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6301 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6361 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6421 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6481 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6541 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6601 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6661 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6721 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6781 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6841 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6901 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6961 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7021 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7081 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcg
//
