LOCUS       sequence-484254-        6561 bp    DNA     circular SYN 21-AUG-2025
DEFINITION  Mammalian vector for transient expression of MCP-VPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6561)
  TITLE     Metabolite-responsive scaffold RNAs for dynamic CRISPR 
            transcriptional regulation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6561)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 21, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pHS-MCP-VPR
FEATURES             Location/Qualifiers
     source          1..6561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   75..109
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        413..792
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        793..996
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     947..967
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1003..1012
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1387..1536
                     /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             1543..1563
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1993..2364
                     /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
                     EDFSSIADMDFSALLSQISS"
     CDS             2383..2952
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     misc_feature    3001..3589
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3054..3074)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3472..3483)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3628..3647)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3682..3701
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3774..3794)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3774..3792)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3813..3829)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3813..3829)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3826..3848)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3837..3853
                     /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(3861..3867,3868..3885,3886..3891))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3906..3927
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4044..4061)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4215..4803)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4295..4314)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4974..5765,5766..5834))
                     /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     5597..5616
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5835..5939)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5965..6420)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6102..6123)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6314..6333
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(6547..6561,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(6561,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttggggcgcg
       61 ccattctaga ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa
      121 tattgctctc tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc
      181 gccgcttgga gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac
      241 tataacgacc gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt
      301 aactcatacg ataattatat tgttatttca tgttctactt acgtgataac ttattatata
      361 tatattttct tgttatagat atcaactaga atgctagcat gggcccagat ctgacattga
      421 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      481 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      541 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      601 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      661 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      721 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      781 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      841 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      901 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      961 aggcgtgtac ggtgggaggt ctatataagc agagctgaat tcgccaccat ggcttcaaac
     1021 ttcacgcagt tcgttcttgt ggataatggc ggcactggag acgttacagt ggctcctagc
     1081 aatttcgcga acgggatcgc agaatggatt agctcaaata gtcgatctca ggcttacaaa
     1141 gtgacatgtt ccgtacggca gtcctccgcg caaaacagga aatataccat caaagtggag
     1201 gtcccgaaag gcgcctggcg atcctacctg aacatggagc tgaccattcc tattttcgcc
     1261 actaactctg attgcgagct gatcgtaaag gctatgcagg gcctgctgaa ggatggcaat
     1321 cctataccaa gcgccattgc tgcaaatagt ggcatctatg aattctcaag cgctgcaggt
     1381 actagtgatg ctttagacga ttttgactta gatatgcttg gttcagacgc gttagacgac
     1441 ttcgacctag acatgttagg ctcagatgca ttggacgact tcgatttaga tatgttgggc
     1501 tccgatgccc tagatgactt tgatctagat atgctaggta gtcccaaaaa gaagaggaaa
     1561 gtgggatccc agtatctgcc cgacacagat gatagacacc gaatcgaaga gaaacgcaag
     1621 cgaacgtatg aaaccttcaa atcgatcatg aagaaatcgc ccttctcggg tccgaccgat
     1681 cccaggcccc caccgagaag gattgcggtc ccgtcccgct cgtcggccag cgtgccgaag
     1741 cctgcgccgc agccctaccc cttcacgtcg agcctgagca caatcaatta tgacgagttc
     1801 ccgacgatgg tgttcccctc gggacaaatc tcacaagcct cggcgctcgc accagcgcct
     1861 ccccaagtcc ttccgcaagc gcctgcccca gcgcctgcac cggcaatggt gtccgccctc
     1921 gcacaggccc ctgcgcccgt ccccgtgctc gcgcctggac cgccccaggc ggtcgctcca
     1981 ccggctccga agccgacgca ggccggagag ggaacactct ccgaagcact tcttcaactc
     2041 cagtttgatg acgaggatct tggagcactc cttggaaact cgacagaccc tgcggtgttt
     2101 accgacctcg cgtcagtaga taactccgaa tttcagcagc ttttgaacca gggtatcccg
     2161 gtcgcgccac atacaacgga gcccatgttg atggaatacc ccgaagcaat cacgagactt
     2221 gtgacgggag cgcagcggcc tcccgatccc gcacccgcac ctttgggggc acctggcctc
     2281 cctaacggac ttttgagcgg cgacgaggat ttctcctcca tcgccgatat ggatttctca
     2341 gccttgctgt cacagatttc cagcggctct ggcagcggca gccgggattc cagggaaggg
     2401 atgtttttgc cgaagcctga ggccggctcc gctattagtg acgtgtttga gggccgcgag
     2461 gtgtgccagc caaaacgaat ccggccattt catcctccag gaagtccatg ggccaaccgc
     2521 ccactccccg ccagcctcgc accaacacca accggtccag tacatgagcc agtcgggtca
     2581 ctgaccccgg caccagtccc tcagccactg gatccagcgc ccgcagtgac tcccgaggcc
     2641 agtcacctgt tggaggatcc cgatgaagag acgagccagg ctgtcaaagc ccttcgggag
     2701 atggccgata ctgtgattcc ccagaaggaa gaggctgcaa tctgtggcca aatggacctt
     2761 tcccatccgc ccccaagggg ccatctggat gagctgacaa ccacacttga gtccatgacc
     2821 gaggatctga acctggactc acccctgacc ccggaattga acgagattct ggataccttc
     2881 ctgaacgacg agtgcctctt gcatgccatg catatcagca caggactgtc catcttcgac
     2941 acatctctgt tttgaattta aatgtttaaa ccatgcaagc ttgatatcaa gcttatcgat
     3001 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3061 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3121 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3181 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3241 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3301 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3361 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     3421 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3481 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3541 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tccttgttta
     3601 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3661 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3721 ggatcctgca ggtatacgcg tatgcggccg cttaattaat ccagcttttg ttccctttag
     3781 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3841 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3901 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3961 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4021 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4081 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4141 aacgcaggaa agaacatgtg agcaaaaggt cagcaaaagg ccaggaaccg taaaaaggcc
     4201 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4261 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4321 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4381 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4441 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4501 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4561 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4621 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4681 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4741 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4801 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4861 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4921 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4981 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5041 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5101 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5161 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5221 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5281 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5341 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5401 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5461 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5521 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5581 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5641 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5701 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5761 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5821 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5881 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5941 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     6001 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6061 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     6121 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     6181 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     6241 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     6301 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     6361 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     6421 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     6481 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     6541 ggttttccca gtcacgacgt t
//
