LOCUS       sequence-321518-        6304 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Constitutive expression of VPR-ZF43(RAAA) under the CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6304)
  AUTHORS   Donahue PS, Draut JW, Muldoon JJ, Edelstein HI, Bagheri N, Leonard 
            JN
  TITLE     The COMET toolkit for composing customizable genetic programs in 
            mammalian cells.
  JOURNAL   Nat Commun. 2020 Feb 7;11(1):779. doi: 10.1038/s41467-019-14147-5.
  PUBMED    32034124
REFERENCE   2  (bases 1 to 6304)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2020-02-7"; volume: "11(1):779. doi"; pages: " 
            10.1038/s41467-019-14147-5"
COMMENT     Sequence Label: pPD385 VPR-ZF43(RAAA)
FEATURES             Location/Qualifiers
     source          1..6304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..975
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             982..1002
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1027..1176
                     /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             1633..1989
                     /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             2008..2577
                     /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"
     primer_bind     complement(2934..2951)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2940..3164
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3210..3638
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3297..3316)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3507..3528
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3661..3857
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3708..3843
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3770..3789
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3977..4098
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4014..4033)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4068..4087
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4147..4163)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4147..4163)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4160..4182)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4171..4187
                     /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(4195..4201,4202..4219,4220..4225))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4240..4261
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4378..4395)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4549..5137)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4629..4648)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5308..6099,6100..6168))
                     /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     5931..5950
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6169..6273)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagtcccaa gctggctagc
      901 gccaccatgg attataagga tcacgatggc gactacaagg accacgatat tgactacaaa
      961 gacgatgacg ataaaatggc tcctaagaaa aagcgcaaag tcggtatcca tggcgttccc
     1021 ggatcagacg cattggacga ttttgatctg gatatgctgg gaagtgacgc cctcgatgat
     1081 tttgaccttg acatgcttgg ttcggatgcc cttgatgact ttgacctcga catgctcggc
     1141 agtgacgccc ttgatgattt cgacctggac atgctgatta actctagaag ttccggatct
     1201 gttggtagcc agtacctgcc cgacaccgac gaccggcacc ggatcgagga aaagcggaag
     1261 cggacctacg agacattcaa gagcatcatg aagaagtccc ccttcagcgg ccccaccgac
     1321 cctagacctc cacctagaag aatcgccgtg cccagcagat ccagcgccag cgtgccaaaa
     1381 cctgcccccc agccttaccc cttcaccagc agcctgagca ccatcaacta cgacgagttc
     1441 cctaccatgg tgttccccag cggccagatc tctcaggcct ctgctctggc tccagcccct
     1501 cctcaggtgc tgcctcaggc tcctgctcct gcaccagctc cagccatggt gtctgcactg
     1561 gctcaggcac cagcacccgt gcctgtgctg gctcctggac ctccacaggc tgtggctcca
     1621 ccagccccta aacctacaca ggccggcgag ggcacactgt ctgaagctct gctgcagctg
     1681 cagttcgacg acgaggatct gggagccctg ctgggaaaca gcaccgatcc tgccgtgttc
     1741 accgacctgg ccagcgtgga caacagcgag ttccagcagc tgctgaacca gggcatccct
     1801 gtggcccctc acaccaccga gcccatgctg atggaatacc ccgaggccat cacccggctc
     1861 gtgacaggcg ctcagaggcc tcctgatcca gctcctgccc ctctgggagc accaggcctg
     1921 cctaatggac tgctgtctgg cgacgaggac ttcagctcta tcgccgatat ggatttctca
     1981 gccttgctgg gctctggcag cggcagccgg gattccaggg aagggatgtt tttgccgaag
     2041 cctgaggccg gctccgctat tagtgacgtg tttgagggcc gcgaggtgtg ccagccaaaa
     2101 cgaatccggc catttcatcc tccaggaagt ccatgggcca accgcccact ccccgccagc
     2161 ctcgcaccaa caccaaccgg tccagtacat gagccagtcg ggtcactgac cccggcacca
     2221 gtccctcagc cactggatcc agcgcccgca gtgactcccg aggccagtca cctgttggag
     2281 gatcccgatg aagagacgag ccaggctgtc aaagcccttc gggagatggc cgatactgtg
     2341 attccccaga aggaagaggc tgcaatctgt ggccaaatgg acctttccca tccgccccca
     2401 aggggccatc tggatgagct gacaaccaca cttgagtcca tgaccgagga tctgaacctg
     2461 gactcacccc tgaccccgga attgaacgag attctggata ccttcctgaa cgacgagtgc
     2521 ctcttgcatg ccatgcatat cagcacagga ctgtccatct tcgacacatc tctgttttct
     2581 ggttctggtt ctggttcagg atccggtacc ggtggagcta gacccgggga gcgccccttc
     2641 cagtgtcgca tttgcatggc gaacttttcg cgccaggaca ggcttgacag gcatacccgt
     2701 actcataccg gtgaaaaacc gtttcagtgt cggatctgta tggcaaattt ctcccagaag
     2761 gagcacttgg cggggcatct acgtacgcac accggcgaga agccattcca atgccgaata
     2821 tgcatggcca acttcagtcg ccgcgacaac ctgaaccggc acctaaaaac ccacctgaga
     2881 ggatcttaag cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2941 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3001 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3061 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3121 ggaagtcaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3181 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3241 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3301 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3361 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3421 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3481 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3541 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3601 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3661 ttaggctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3721 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3781 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3841 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga ggttgggctt
     3901 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     3961 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4021 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4081 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4141 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4201 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4261 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4321 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4381 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4441 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4501 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4561 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4621 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4681 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4741 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4801 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4861 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4921 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4981 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5041 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5101 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5161 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5221 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5281 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5341 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5401 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag atccacgctc
     5461 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5521 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5581 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5641 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5701 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5761 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5821 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5881 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5941 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6001 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6061 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6121 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6181 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6241 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6301 cgtc
//
