LOCUS       sequence-420428-        6920 bp    DNA     circular SYN 15-APR-2025
DEFINITION  dCas9-VP64 expressed under control of TRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6920)
  AUTHORS   Nguyen et al.
  TITLE     Implantation of engineered adipocytes that outcompete tumors for 
            resources suppresses cancer progression
  JOURNAL   bioRxiv 2023.03.28.534564
REFERENCE   2  (bases 1 to 6920)
  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: "bioRxiv 
            2023.03.28.534564"
COMMENT     Sequence Label: TRE-dCas9(sa)-VP64-AAV
FEATURES             Location/Qualifiers
     source          1..6920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     polyA_signal    793..1000
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     989..1006
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1040..1189)
                     /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             complement(1220..1267)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(4448..4468)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4471..4480
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4482..4520)
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    join(4553..4571,4572..4594,4595..4613,4614..4636,4637..4655,
                     4656..4678,4679..4697,4698..4720,4721..4739,4740..4762,
                     4763..4781,4782..4804,4805..4823)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     repeat_region   4833..4973
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4833..4962
                     /label=AAV2 ITR
     rep_origin      5048..5503
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5135..5154)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5345..5366
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5520..5539)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5639..5661
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5699..5717)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5785..5889
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5890..5958,5959..6750)
                     /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     complement(6108..6127)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggcctcta gctccccagc atgcctgcta ttctcttccc aatcctcccc cttgctgtcc
      841 tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc
      901 tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg
      961 gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcgaggctga tcagcgagct
     1021 ctaggaattc tctagatcac agcatgtcca ggtcgaaatc atcaagggcg tcactgccga
     1081 gcatgtcgag gtcaaagtca tcaagggcat ccgaaccaag catgtcaagg tcaaaatcat
     1141 cgagggcgtc acttcccagc atatccagat caaaatcgtc caatgcgtca gcccgtccgg
     1201 aaccgctggc ctcggatccc tttttctttt ttgcctggcc ggcctttttc gtggccgccg
     1261 gccttttgcc ctttttgatg atctgagggt gcttcttaga tttcacttca tacaggttgc
     1321 ccagaatgtc tgtgctgtac ttcttaatgc tctgggtctt ggaggcgatt gtcttaatga
     1381 tcctgggggg cctcttgtcg ttcatgtttt ccaggtactc gcggtaggtg atgtcgatca
     1441 tgttcacttc gatccggttc agcaggtcgt tgttcacgcc gatcactcta tacagctcgc
     1501 cgttgatctt gatcagatcg ttgttgtaga aggaggcgat aaactcggcc tggttgctga
     1561 tcttcttcag cttcttagct tcctcatagc acttgctatt cacttcgtag tagttttctt
     1621 ttttgatcac atccagattc ttcacggtca cgaacttgta cacgccattg tccaggtaca
     1681 cgtcgaatct gtagggcttc agggacagct tcacgacctt gtttctgctg ttggggtagt
     1741 cgtcggtgat gtccagatgg gcgttcagtt tgttgccgta atacttaatc ttcttgatca
     1801 cggggccgtt gtcctttttg gagtacttgg tcaggtagtt cccggtttcc tcgtagtact
     1861 tgtacagggg attcttctcg tcgccgtact gttccataat cagcttcagt ttctggtagg
     1921 tctgggggtc gtggtggtac atcagcagct tttcggggct cttgttgatc agctttttca
     1981 gcttgtcatt gtccttgtcg tacaggccgt tcagattgtt cacgatcagg gtgttgccct
     2041 tgtcgtcctt ccgggtggag tacagggtgt cgttaatcag ctctctatta ggcttcttgt
     2101 ccacccggtg gctgtacttg tagtccttga agtccttaat gtgcttgatc tggtgggggg
     2161 tgatgaagat ctctttgtac tcctgctcgg tttcgatctc gggcatgctc tcggcctgct
     2221 tttcctcgaa catctggttt tccatcactt ttttggcctt gtccagtttc ttccactctt
     2281 tgaagatgaa atcggcgttg gcaatgatca gggcgtcctc ggcgtggtgc ttgtacccct
     2341 tgttccgctc tttcttaaac ttccacttcc gccgcagaaa gctggtgaag ccgccattga
     2401 tggacttcac tttcacgtcc aggttgttca ctctgaagta gctccgcagc aggttcatca
     2461 ggcctctggt ggcgtatctg gtatccacca ggttccggtt gatgaagtct ttctgcacgg
     2521 agaacctgtt gatgtcccgt tcttccagca gatactcttt cttggtcttg ctgattctgc
     2581 ccttgccctt ggccagattc aggatgtgct tcttgaaggt ttcgtagctg atcttgctgt
     2641 cgctgctgct caggtactgg aatggggtcc ggttgccctt cttgctggct tcttcctgct
     2701 tcacgagcac cttgttgttg aagctgttgt cgaaggacac gcttctgggg atgatgtggt
     2761 ccacctcata gttgaagggg ttgttcagca gatcttccag agggatggct tccaggctgt
     2821 acaggcactt gccttcctgc atgtcgtgca gcttgatctt ctcgatcagg tacttggcgt
     2881 tctctttgcc ggtggtccgg atgatttcct cgatccgctc gttggtctgc cggttccgct
     2941 tctgcatctc gttgatcatt ttctgggcgt ccttggagtt cttctcgcgg gccagctcga
     3001 taatgatgtc gttgggcagg ccgtacttct tgatgatggc gttgatcact ttgatgctct
     3061 ggatgaagct tctcttcacg acggggctca ggatgaagtc gtccaccagg gtggtgggga
     3121 tctctttctg ctgggacagg tccaccttct tgggcaccag cttcagccgg ttgaagatag
     3181 cgatctggtt gtcgttggtg tgccacagct cgtccaggat caggttgatg gccttcaggc
     3241 tcaggttgtg ggtgccggta tagcccttca gattagagat ctgctcgatc tcttcctggg
     3301 tcagctcgga gttcagattg gtcagttctt cctggatgtc ctcgctgctc tggtagatgg
     3361 tcaggatctt ggcaatctga tccagcagct cggcgttctc aataatctct ttccgggcgg
     3421 taatgtcctt gatgtcgtgg tacaccttca ggttggtgaa ctcgggcttg ccggtgctgg
     3481 tcactctgta gcccttaata tcctcttcgt tcacgaggat ttctttggcg atctgcttca
     3541 gggtgggctt cttcttctgc ttgaacacgt tctcgatgat ctggaacttc tcgtaatatt
     3601 ccagcttctc gttctcgtcc ctggtgatca cgagattgtt caggtcgttc agggcgttgt
     3661 acaggtcggc gttgtaggcg tacttcacgc tccgcagttc ctcggggaag taggtgcagt
     3721 ggcccatcag catctcgtac cattctttga tgtccttcca gccgaagggg ctgccctcgc
     3781 caggtccctc atagtaggtc cgccgggttt ccagcaggtc gatgtaggtg tcgatgaagc
     3841 tctggtccag ctggtggtag gccttctgca ccttcagcag ctgtttggct tctttcacgt
     3901 agtcgctggt cttgaatctg ttgatgctgc cccgcacttc gccgtctttc ttcagccgtt
     3961 ccagctgcag ttcggccacg tatttctctt ccagggcctt gctgttccgg ctgatctgct
     4021 ctttggtgga cagctcgttg ccggtgtcct cttccacctc gttcacgttg tgcacgcctc
     4081 ttctcttggc caggtgcagc agggcggcag agaactcttc ctcgctcagc ttctggctca
     4141 ggcccttcac tctggcctcg taggggttga tgccgctcag ctcgctgtgg tcggtcagca
     4201 ggttgtagtc gaacagcagc ttcttcactc tctggattct atgccgcctc cgccgcttca
     4261 gccttctggc gcctctcttg ctccgcctgc cctcgttgtt ttccacgttg gcctctttga
     4321 acagccgcac gccggcatcg atcacgtccc gtgtctcgta gtcgatgatg ccgtagccca
     4381 cgctggtgat gccgatggcc aggcccagga tgtagttccg cttggctgct gggactccgt
     4441 ggataccgac cttccgcttc ttctttgggg ccatggtggc aagctctgct tatataggcc
     4501 tcccaccgta cacgcctacc tcgacccggg taccgagctc gactttcact tttctctatc
     4561 actgataggg agtggtaaac tcgactttca cttttctcta tcactgatag ggagtggtaa
     4621 actcgacttt cacttttctc tatcactgat agggagtggt aaactcgact ttcacttttc
     4681 tctatcactg atagggagtg gtaaactcga ctttcacttt tctctatcac tgatagggag
     4741 tggtaaactc gactttcact tttctctatc actgataggg agtggtaaac tcgactttca
     4801 cttttctcta tcactgatag ggaggcggcc gcaggaaccc ctagtgatgg agttggccac
     4861 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4921 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4981 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     5041 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     5101 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     5161 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5221 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     5281 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5341 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     5401 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     5461 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     5521 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     5581 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5641 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5701 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5761 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5821 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5881 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5941 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     6001 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     6061 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     6121 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     6181 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     6241 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6301 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6361 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6421 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6481 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6541 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6601 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6661 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6721 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6781 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6841 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6901 gaaaagatca aaggatcttc
//
