LOCUS       sequence-497385-        7137 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Expression of PKA T128K mutant (PRKACAE128K).
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 E128K
FEATURES             Location/Qualifiers
     source          1..7137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(101..120)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      914..1502
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1403..1422
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1656..1673
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1790..1811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1826..1831,1832..1849,1850..1856)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1864..1880
                     /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     1869..1891
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1888..1904
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1888..1904
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1953..2074
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1964..1983)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2018..2037
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2248..3042)
                     /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(2359..2378)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2969..2988
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3109..3438)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3123..3258
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3177..3196)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3423..3443
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3452..3880)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3562..3583)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3774..3793
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3926..4150
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4139..4156
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        4176..4194
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4176..4193
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(4246..4272)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      5328..5337
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5404..6586)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5413..6355
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(5436..5456)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     6817..6836
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6915..7019
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7020..7088,7089..7137,1..743)
                     /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 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
       61 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
      121 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
      181 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
      241 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
      301 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
      361 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
      421 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
      481 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
      541 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
      601 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
      661 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
      721 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
      781 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
      841 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
      901 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      961 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1021 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     1081 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     1141 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     1201 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     1261 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     1321 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     1381 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     1441 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     1501 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     1561 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     1621 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     1681 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     1741 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     1801 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     1861 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     1921 tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt ttggacaaac
     1981 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     2041 atttgtaacc attataagct gcaataaaca agttggggtg ggcgaagaac tccagcatga
     2101 gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc
     2161 tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt
     2221 cggtcatttc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc
     2281 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
     2341 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
     2401 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
     2461 cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt
     2521 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
     2581 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
     2641 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
     2701 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
     2761 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
     2821 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc
     2881 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
     2941 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
     3001 agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc
     3061 tgtctcttga tcagatccga aaatggatat acaagctccc gggagctttt tgcaaaagcc
     3121 taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggcag aggcggcctc
     3181 ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg
     3241 cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag
     3301 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc
     3361 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     3421 acaccctaac tgacacacat tccacagaat taattcgcgt taaatttttg ttaaatcagc
     3481 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     3541 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     3601 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     3661 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     3721 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3781 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3841 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc cctagagccc
     3901 cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc atgcctgcta
     3961 ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga
     4021 cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg
     4081 gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta
     4141 gaaggcacag tcgaggctga tcagcgagct ctagcattta ggtgacacta tagaataggg
     4201 ccctctagag gccctggagg ccgatatctc gagtgcggcc gcctaggcgt aatctggcac
     4261 gtcgtatggg tagctagcaa actcagaaaa ctccttgcca cacttctcat tgatggagac
     4321 ccggatttct tcttcctcat agtcgtcaaa gttactcgta tccccagggc ctttaaactt
     4381 tggtatgaag ggagcttcca ccttcctctg gtagatggca atccagtcag ttgtggcaaa
     4441 ccacttgtgg ttcttgatat cgttgacccc attcttgagg ttcccaaagc gcttggtgag
     4501 atctacctgc aggaggttcc gcagcaggtc cttcaagtca gagctgaagt gggaagggaa
     4561 gcgcaccttc ccagagacga tcttctcata gatctggatg ggctggtctg cgaagaaggg
     4621 cgggtagcca gcggccattt catagataag aacccccagg gcccaccagt ccacggcctt
     4681 gttgtagcct ttgctcagga taatctcagg ggccaggtac tcaggggtgc cgcacaaggt
     4741 ccaagtgcgg cccttcacgc gcttggcgaa accgaagtct gtcacctgaa tgtagccctg
     4801 ctggtcaatg agcagattct ccggcttcag gtccctgtag atgagatcca gcgagtgcag
     4861 atactcaaag gtcaggacga tctgggccgc gtagaaacgg gcatggggct cactgaacct
     4921 tccgatccgc cgtaggtgtg agaacatctt cccgccgggc acgtactcca tgaccatgta
     4981 taagtttgag ttgtccttga aggagaactc gagtttgacg aggaacggaa agttgacagc
     5041 ttgcaggatg cgcttttcat tcagggtgtg ttcgatctgt ttcagtttca ccaccttctg
     5101 tttgtcgagg atcttcatgg catagtggtt cccggtctcc ttgtgtttca ccagcatcac
     5161 ccgcccgaag gagcccgtgc cgagggtctt gattcgttca aactgatcca agtgggctgt
     5221 gttctgagcg ggactttccc attttttaag aaaatcttct ttggctttgg ctaagaattc
     5281 tttcacgctc tcctgctcgc tgcccttctt ggcggcggcg gcgttgccca tggtggcggc
     5341 ggatccagat ctggtaccaa gcttgatggc cacaatggcc gttaactgag cttcgccaat
     5401 tcctcacgac acctgaaatg gaagaaaaaa actttgaacc actgtctgag gcttgagaat
     5461 gaaccaagat ccaaactcaa aaagggcaaa ttccaaggag aattacatca agtgccaagc
     5521 tggcctaact tcagtctcca cccactcagt gtggggaaac tccatcgcat aaaacccctc
     5581 cccccaacct aaagacgacg tactccaaaa gctcgagaac taatcgaggt gcctggacgg
     5641 cgcccggtac tccgtggagt cacatgaagc gacggctgag gacggaaagg cccttttcct
     5701 ttgtgtgggt gactcacccg cccgctctcc cgagcgccgc gtcctccatt ttgagctccc
     5761 tgcagcaggg ccgggaagcg gccatctttc cgctcacgca actggtgccg accgggccag
     5821 ccttgccgcc cagggcgggg cgatacacgg cggcgcgagg ccaggcacca gagcaggccg
     5881 gccagcttga gactaccccc gtccgattct cggtggccgc gctcgcaggc cccgcctcgc
     5941 cgaacatgtg cgctgggacg cacgggcccc gtcgccgccc gcggccccaa aaaccgaaat
     6001 accagtgtgc agatcgatct tggcccgcat ttacaagact atcttgccag aaaaaaagcg
     6061 tcgcagcagg tcatcaaaaa ttttaaatgg ctagagactt atcgaaagca gcgagacagg
     6121 cgcgaaggtg ccaccagatt cgcacgcggc ggccccagcg cccaggccag gcctcaactc
     6181 aagcacgagg cgaaggggct ccttaagcgc aaggcctcga actctcccac ccacttccaa
     6241 cccgaagctc gggatcaaga atcacgtact gcagccaggt ggaagtaatt caaggcacgc
     6301 aagggccata acccgtaaag aggccaggcc cgcgggaacc acacacggca cttacctgtg
     6361 ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc gactactgca cttatatacg
     6421 gttctccccc accctcggga aaaaggcgga gccagtacac gacatcactt tcccagttta
     6481 ccccgcgcca ccttctctag gcaccggttc aattgccgac ccctcccccc aacttctcgg
     6541 ggactgtggg cgatgtgcgc tctgcccact gacgggcacc ggagccaatt cccactcctt
     6601 tcaagaccta gaaggtccat tagctgcaaa gattcctctc tgtttaaaac tttatccatc
     6661 tttgcaaagc tcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaattgt
     6721 cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca
     6781 acacacaagc agggagcaga tactggctta actatgcggc atcagagcag attgtactga
     6841 gagtgcacca taggggatcg ggagatcgat ctcccgatcc gtcgacgtca ggtggcactt
     6901 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     6961 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     7021 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     7081 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtg
//
