LOCUS       sequence-254859-        7615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for the expression of mRuby2-fused human PRKACA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7615)
  AUTHORS   Fonseca JP, Weinberg ZY, Aslankoohi E, El-Samad H
  TITLE     Biphasic Response of Protein Kinase A to Cyclic Adenosine 
            Monophosphate Triggers Distinct Epithelial Phenotypes
  JOURNAL   bioRxiv 2020, 747030
REFERENCE   2  (bases 1 to 7615)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2020, 747030"
COMMENT     Sequence Label: pJPF0190
FEATURES             Location/Qualifiers
     source          1..7615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..35
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     intron          595..1612
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1536..1558
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1620..1639
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2735..3442
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     primer_bind     complement(3457..3477)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3462..3791
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3642..3777
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3801..4400
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3801..3820)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4297..4317
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     repeat_region   4641..4703
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4754..4774)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4754..4772)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4793..4809)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4793..4809)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4806..4828)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4817..4833
                     /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(4841..4847,4848..4865,4866..4871))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4886..4907
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5024..5041)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5195..5783)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5275..5294)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5954..6745,6746..6814))
                     /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     6577..6596
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6815..6919)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6945..7400)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7082..7103)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7294..7313
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7527..7549
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7541..7558
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7542..7558
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7568..7587
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7568..7586
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt
       61 tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg cttggagctc
      121 ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata acgaccgcgt
      181 gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact catacgataa
      241 ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt
      301 atagatatca actagaatgc tagcatgggc ccagatctcc atggtcgagg tgagccccac
      361 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
      421 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggcgaggg gcggggcggg
      481 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
      541 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
      601 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
      661 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
      721 tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt
      781 gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt
      841 gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg
      901 cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt
      961 gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg
     1021 ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aacccccccc tgcacccccc
     1081 tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc
     1141 ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc
     1201 gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg
     1261 tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg
     1321 acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1381 tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct
     1441 tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg
     1501 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     1561 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     1621 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattcatgg gcaacgccgc
     1681 cgccgccaag aagggcagcg agcaggagag cgtgaaagaa ttcttagcca aagccaaaga
     1741 agattttctt aaaaaatggg aaagtcccgc tcagaacaca gcccacttgg atcagtttga
     1801 acgaatcaag accctcggca cgggctcctt cgggcgggtg atgctggtga aacacaagga
     1861 gaccgggaac cactatgcca tgaagatcct cgacaaacag aaggtggtga aactgaaaca
     1921 gatcgaacac accctgaatg aaaagcgcat cctgcaagct gtcaactttc cgttcctcgt
     1981 caaactcgag ttctccttca aggacaactc aaacttatac atggtcatgg agtacgtgcc
     2041 cggcggggag atgttctcac acctacggcg gatcggaagg ttcagtgagc cccatgcccg
     2101 tttctacgcg gcccagatcg tcctgacctt tgagtatctg cactcgctgg atctcatcta
     2161 cagggacctg aagccggaga atctgctcat tgaccagcag ggctacattc aggtgacaga
     2221 cttcggtttc gccaagcgcg tgaagggccg cacttggacc ttgtgcggca cccctgagta
     2281 cctggcccct gagattatcc tgagcaaagg ctacaacaag gccgtggact ggtgggccct
     2341 gggggttctt atctatgaaa tggccgctgg ctacccgccc ttcttcgcag accagcccat
     2401 ccagatctat gagaagatcg tctctgggaa ggtgcgcttc ccttcccact tcagctctga
     2461 cttgaaggac ctgctgcgga acctcctgca ggtagatctc accaagcgct ttgggaacct
     2521 caagaatggg gtcaacgata tcaagaacca caagtggttt gccacaactg actggattgc
     2581 catctaccag aggaaggtgg aagctccctt cataccaaag tttaaaggcc ctggggatac
     2641 gagtaacttt gacgactatg aggaagaaga aatccgggtc tccatcaatg agaagtgtgg
     2701 caaggagttt tctgagtttt tgggtaccga attcgtgtct aagggcgaag agctgatcaa
     2761 ggagaatatg cgtatgaagg tggtcatgga aggttcggtc aacggccacc aattcaaatg
     2821 cacaggtgaa ggagaaggca atccgtacat gggaactcaa accatgagga tcaaagtcat
     2881 cgagggagga cccctgccat ttgcctttga cattcttgcc acgtcgttca tgtatggcag
     2941 ccgtactttt atcaagtacc cgaaaggcat tcctgatttc tttaaacagt cctttcctga
     3001 gggttttact tgggaaagag ttacgagata cgaagatggt ggagtcgtca ccgtcatgca
     3061 ggacaccagc cttgaggatg gctgtctcgt ttaccacgtc caagtcagag gggtaaactt
     3121 tccctccaat ggtcccgtga tgcagaagaa gaccaagggt tgggagccta atacagagat
     3181 gatgtatcca gcagatggtg gtctgagggg atacactcat atggcactga aagttgatgg
     3241 tggtggccat ctgtcttgct ctttcgtaac aacttacagg tcaaaaaaga ccgtcgggaa
     3301 catcaagatg cccggtatcc atgccgttga tcaccgcctg gaaaggttag aggaaagtga
     3361 caatgaaatg ttcgtagtac aacgcgaaca cgcagttgcc aagttcgccg ggcttggtgg
     3421 tgggatggac gagctgtaca agtaagtcga ccctgtggaa tgtgtgtcag ttagggtgtg
     3481 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3541 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3601 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3661 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3721 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3781 gcttttgcaa aaagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     3841 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     3901 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     3961 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     4021 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     4081 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     4141 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     4201 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     4261 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     4321 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     4381 cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     4441 cacgacccca tgcatcgata ctagttaaaa gttttgttac tttatagaag aaattttgag
     4501 tttttgtttt tttttaataa ataaataaac ataaataaat tgtttgttga atttattatt
     4561 agtatgtaag tgtaaatata ataaaactta atatctattc aaattaataa ataaacctcg
     4621 atatacagac cgataaaaca catgcgtcaa ttttacgcat gattatcttt aacgtacgtc
     4681 acaatatgat tatctttcta gggttaatct agtatacgcg tatgcggccg cttaattaat
     4741 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     4801 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4861 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4921 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4981 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5041 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5101 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5161 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5221 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5281 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5341 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5401 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5461 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5521 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5581 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5641 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5701 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5761 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5821 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5881 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5941 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6001 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6061 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6121 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6181 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6241 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6301 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6361 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6421 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6481 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6541 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6601 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6661 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6721 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6781 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6841 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6901 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     6961 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     7021 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     7081 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     7141 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     7201 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     7261 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     7321 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     7381 tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg
     7441 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     7501 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     7561 gcgcgcgtaa tacgactcac tatagggcga attggggcgc gccattctag attaa
//
