LOCUS       sequence-292002-        6138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive PGK driven expression of intein with miniVPR for 3-way 
            split activator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6138)
  AUTHORS   Kempton HR, Goudy LE, Love KS, Qi LS
  TITLE     Multiple Input Sensing and Signal Integration Using a Split Cas12a 
            System.
  JOURNAL   Mol Cell. 2020 Jan 30. pii: S1097-2765(20)30037-X. doi: 
            10.1016/j.molcel.2020.01.016.
  PUBMED    32027839
REFERENCE   2  (bases 1 to 6138)
  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: "Mol Cell. 
            2020 Jan 30. pii: S1097-2765(20)30037-X. doi: 
            10.1016/j.molcel.2020.01.016."
COMMENT     Sequence Label: pSLQ8637 PB-PGK-C intein-2XNLS-miniVPR-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..6138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..500
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(36..59)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     433..452
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             628..648
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             655..675
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             712..861
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             868..888
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1021..1377
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             1612..2322
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1757..1775)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2025..2044
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(2341..2359)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2342..2359)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2379..2396)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2385..2609
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2825..2887
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2938..2958)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2938..2956)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2977..2993)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2977..2993)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2990..3012)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3001..3017
                     /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(3025..3031,3032..3049,3050..3055))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3070..3091
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3208..3225)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3379..3967)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3459..3478)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4138..4929,4930..4998))
                     /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     4761..4780
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4999..5103)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5129..5584)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5266..5287)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5478..5497
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5711..5733
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5725..5742
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5726..5742
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5752..5771
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5752..5770
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   5800..5834
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
       61 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
      121 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
      181 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
      241 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
      301 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
      361 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
      421 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
      481 cctcatctcc gggcctttcg agaattcggt accatgataa agatcgccac tcggaagtat
      541 ttggggaaac agaacgtgta tgatattggt gtcgagagag accacaactt tgcacttaag
      601 aatgggttca tagcgtctag cggctccccc aagaaaaaac gcaaggtgga agatcctaag
      661 aaaaagcgga aagtggacgg cattggtagt gggagcaacg gcagcagcct cgatgcttta
      721 gacgattttg acttagatat gcttggttca gacgcgttag acgacttcga cctagacatg
      781 ttaggctcag atgcattgga cgacttcgat ttagatatgt tgggctccga tgccctagat
      841 gactttgatc tagatatgct aggtagtccc aaaaagaaga ggaaagtggg atccgtcctt
      901 ccgcaagcgc ctgccccagc gcctgcaccg gcaatggtgt ccgccctcgc acaggcccct
      961 gcgcccgtcc ccgtgctcgc gcctggaccg ccccaggcgg tcgctccacc ggctccgaag
     1021 ccgacgcagg ccggagaggg aacactctcc gaagcacttc ttcaactcca gtttgatgac
     1081 gaggatcttg gagcactcct tggaaactcg acagaccctg cggtgtttac cgacctcgcg
     1141 tcagtagata actccgaatt tcagcagctt ttgaaccagg gtatcccggt cgcgccacat
     1201 acaacggagc ccatgttgat ggaatacccc gaagcaatca cgagacttgt gacgggagcg
     1261 cagcggcctc ccgatcccgc acccgcacct ttgggggcac ctggcctccc taacggactt
     1321 ttgagcggcg acgaggattt ctcctccatc gccgatatgg atttctcagc cttgctgtca
     1381 ggctctggca gcggcagcga cctttcccat ccgcccccaa ggggccatct ggatgagctg
     1441 acaaccacac ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa
     1501 ttgaacgaga ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc
     1561 agcacaggac tgtccatctt cgacacatct ctgtttggca gcagcggatc catggtgagc
     1621 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
     1681 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     1741 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     1801 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
     1861 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     1921 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     1981 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     2041 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     2101 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     2161 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     2221 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     2281 ggccgccact ccaccggcgg catggacgag ctgtacaagt aactcgagag agggccctat
     2341 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     2401 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2461 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2521 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     2581 gcatgctggg gatgcggtgg gctctatggc ttctgaggtc gaggcggccg ctgatcacaa
     2641 ttgtctagtt aaaagttttg ttactttata gaagaaattt tgagtttttg ttttttttta
     2701 ataaataaat aaacataaat aaattgtttg ttgaatttat tattagtatg taagtgtaaa
     2761 tataataaaa cttaatatct attcaaatta ataaataaac ctcgatatac agaccgataa
     2821 aacacatgcg tcaattttac gcatgattat ctttaacgta cgtcacaata tgattatctt
     2881 tctagggtta atctagtata cgcgtatgcg gccgcttaat taatccagct tttgttccct
     2941 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3001 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3061 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3121 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3181 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3241 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3301 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3361 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3421 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3481 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3541 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3601 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3661 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3721 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3781 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3841 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3901 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3961 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4021 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4081 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4141 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4201 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4261 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4321 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4381 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4441 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4501 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4561 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4621 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4681 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4741 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4801 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4861 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4921 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4981 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5041 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5101 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     5161 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     5221 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     5281 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     5341 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     5401 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     5461 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     5521 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     5581 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     5641 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     5701 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac
     5761 tcactatagg gcgaattggg gcgcgccatt ctagattaac cctagaaaga tagtctgcgt
     5821 aaaattgacg catgcattct tgaaatattg ctctctcttt ctaaatagcg cgaatccgtc
     5881 gctgtgcatt taggacatct cagtcgccgc ttggagctcc cgtgaggcgt gcttgtcaat
     5941 gcggtaagtg tcactgattt tgaactataa cgaccgcgtg agtcaaaatg acgcatgatt
     6001 atcttttacg tgacttttaa gatttaactc atacgataat tatattgtta tttcatgttc
     6061 tacttacgtg ataacttatt atatatatat tttcttgtta tagatatcaa ctagaatgct
     6121 agcatgggcc cagatctc
//
