LOCUS       sequence-455847-        8610 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Adenoviral helper plasmid containing inducible E2A and L4-22k/33k 
            genes and constitutive VA RNA I gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8610)
  AUTHORS   Doshi J, Couto E, Staiti J, Vandenberghe LH, Zabaleta N
  TITLE     E2A, VA RNA I, and L4-22k adenoviral helper genes are sufficient for
            AAV production in HEK293 cells.
  JOURNAL   Mol Ther Methods Clin Dev. 2024 Nov 13;32(4):101376. doi: 
            10.1016/j.omtm.2024.101376. eCollection 2024 Dec 12.
  PUBMED    39670178
REFERENCE   2  (bases 1 to 8610)
  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 Ther 
            Methods Clin Dev."; date: "2024-11-13"; volume: "32(4):101376. doi";
            pages: " 10.1016/j.omtm.2024.101376. eCollection 2024 Dec 12"
COMMENT     Sequence Label: JD179
FEATURES             Location/Qualifiers
     source          1..8610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    95..116
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(131..136,137..154,155..161)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    169..185
                     /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)."
     primer_bind     174..196
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     193..209
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     193..209
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     228..248
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        230..248
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   270..332
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     terminator      complement(723..859)
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     terminator      complement(889..935)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    1050..1171
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      2103..2112
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    join(2310..2328,2329..2351,2352..2370,2371..2393,2394..2412,
                     2413..2435,2436..2454,2455..2477,2478..2496,2497..2519,
                     2520..2538,2539..2561,2562..2580)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     polyA_signal    4458..4682
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4697..4818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(4868..5581,5582..5584,5585..5587))
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     5262..5281
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5521..5542
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      5584..5593
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     repeat_region   5956..5990
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6027..6046)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6028..6046)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6053..6070)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6053..6069)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6062..6084)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(6211..6666)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6348..6369)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6560..6579
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6693..6797
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6798..6866,6867..7658)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSS
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7016..7035)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7829..8417
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8318..8337
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8571..8588
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
       61 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      121 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      181 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      241 actaaaggga acctccccca attccttaac cctagaaaga taatcatatt gtgacgtacg
      301 ttaaagataa tcatgcgtaa aattgacgca tgaggtctga agaggagttt acgtccagcc
      361 aaagctccat ggtcgggacg ctctggccgg tgaggcgtgc gcagtcgttg acgctctaga
      421 ccgtgcaaaa ggagagcctg taagcgggca ctcttccgtg gtctggtgga taaattcgca
      481 agggtatcat ggcggacgac cggggttcga accccggatc cggccgtccg ccgtgatcca
      541 tgcggttacc gcccgcgtgt cgaacccagg tgtgcgacgt cagacaacgg gggagcgctc
      601 cttttggctt ccttccaggc gcggcggctg ctgcgctagc ttttttggcc actggccgcg
      661 cgcggcgtaa gcggttaggc tggatcgaaa tcgagagatc gcctgcagga tagctgacga
      721 ccttaaggat aaatttctgg taaggaggac acgtatggaa gtgggcaagt tggggaagcc
      781 gtatccgttg ctgaatctgg catatgtggg agtataagac gcgcagcgtc gcatcaggca
      841 tttttttctg cgccaatgcc ggtagatagc aatccggccc gaggggcaca aaaaacccct
      901 caagacccgt ttagaggccc caaggggtta tgctacgata ccgatagaga tgggccaata
      961 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     1021 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     1081 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     1141 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tgagctgaag gtacgctgta
     1201 tctcagtcag tcaagctagc ctagtcctta agagtcagcg cgcagtattt actgaagaga
     1261 gcctccgcgt cttccagcgt gcgccgaagc tgatcttcgc ttttgtgata caggcagctg
     1321 cgggtgaggg atcgcagaga cctgtttttt attttcagct cttgttcttg gcccctgctc
     1381 tgttgaaata tagcatacag agtgggaaaa atcctgtttc taagctcgcg ggtcgatacg
     1441 ggttcgttgg gcgccagacg cagcgctcct cctcctgctg ctgccgccgc tgtggatttc
     1501 ttgggctttg tcagagtctt gctatccggt cgcctttgct tctgtgtggc cgctgctgtt
     1561 gctgccgctg ccgccggtgc agtatgggct gtagagatga cggtagtaat gcaggatgtt
     1621 acgggggaag gccacgccgt gatggtagag aagaaagcgg cgggcgaagg agatgttgcc
     1681 cccacagtct tgcaagcaag caactatggc gttcttgtgc ccgcgccatg agcggtagcc
     1741 ttggcgctgt tgttgctctt gggctaacgg cggcggctgc ttggacttac cggccctggt
     1801 tccagtggtg tcccatctac ggttgggtcg gcgaacgggc agtgccggcg gcgcctgagg
     1861 agcggaggtt gtagccatgc tggaaccggt tgccgatttc tggggcgccg gcgaggggaa
     1921 tgcgaccgag ggtgacggtg tttcgtctga cacctcttcg acctcggaag cttcctcgtc
     1981 taggctctcc cagtcttcca tcatgtcctc ctcctcctcg tccaaaacct cctctgcctg
     2041 actgtcccag tattcctcct cgtccgtggg tggcggcggc agctgcagct tctttttggg
     2101 tgccatggtg gccctaccgg tgaacgtttt tgaacgtttc taacgtctct ctggatattt
     2161 cgttctccgc ctcgtgggcg tttttaagct ctaaattgtt agaagattcc tcagccaaga
     2221 ttatggaggt caaaacagcg tggatggcgt ctccaggcga tctgacggtt cactaaacga
     2281 gctctgctta tataggtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc
     2341 gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat
     2401 cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg
     2461 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact
     2521 ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga
     2581 aaagtgaaag tcgagctcgc tggtcgagta tgtcgaggtg gcgtgtacgg tgggaggcct
     2641 atataagcag agctcgttta gtgaaccgtc agatcgcctg gagaattcag atctcagctg
     2701 gtcgacccta ggttgtgata caggcagctg cggagcgttt gccgcgcgtt tctcgtcgcg
     2761 tccaagaccc tcaaagattt ttggcacttc gtcgagcgag gcgatatcag gagaaggaag
     2821 gtaccgccag catggccagt cgggaagagg agcagcgcga aaccaccccc gagcgcggac
     2881 gcggtgcggc gcgacgtcca ccaaccatgg aggacgtgtc gtccccgtcg ccgtcgccgc
     2941 cgcctccccg cgcgccccca aaaaagcggc tgaggcggcg gctcgagtcc gaggacgaag
     3001 aggactcgtc acaagatgcg ctggtgccgc gcacacccag cccgcggcca tcgacctcga
     3061 cggcggattt ggccattgcg tccaaaaaga aaaagaagcg cccctctccc aagcccgagc
     3121 gcccgccatc cccagaggtg atcgtggaca gcgaggaaga aagagaagat gtggcgctac
     3181 aaatggtggg tttcagcaac ccaccggtgc taatcaagca cggcaaggga ggtaagcgca
     3241 cggtgcggcg gctgaatgag gacgacccag tggcgcgggg tatgcggacg caagaggaaa
     3301 aggaagagtc cagtgaagcg gaaagtgaaa gcacggtgat aaacccgctg agcctgccga
     3361 tcgtgtctgc gtgggagaag ggcatggagg ctgcgcgcgc gttgatggac aagtaccacg
     3421 tggataacga tctaaaggca aacttcaagc tactgcctga ccaagtggaa gctctggcgg
     3481 ccgtatgcaa gacctggcta aacgaggagc accgcgggtt gcagctgacc ttcaccagca
     3541 acaagacctt tgtgacgatg atggggcgat tcctgcaggc gtacctgcag tcgtttgcag
     3601 aggtaaccta caagcaccac gagcccacgg gctgcgcgtt gtggctgcac cgctgcgctg
     3661 agatcgaagg cgagcttaag tgtctacacg ggagcattat gataaataag gagcacgtga
     3721 ttgaaatgga tgtgacgagc gaaaacgggc agcgcgcgct gaaggagcag tctagcaagg
     3781 ccaagatcgt gaagaaccgg tggggccgaa atgtggtgca gatctccaac accgacgcaa
     3841 ggtgctgcgt gcatgacgcg gcctgtccgg ccaatcagtt ttccggcaag tcttgcggca
     3901 tgttcttctc tgaaggcgca aaggctcagg tggcttttaa gcagatcaag gctttcatgc
     3961 aggcgctgta tcctaacgcc cagaccgggc acggtcacct tctgatgcca ctacggtgcg
     4021 agtgcaactc aaagcctggg catgcaccct ttttgggaag gcagctacca aagttgactc
     4081 cgttcgccct gagcaacgcg gaggacctgg acgcggatct gatctccgac aagagcgtgc
     4141 tggccagcgt gcaccacccg gcgctgatag tgttccagtg ctgcaaccct gtgtatcgca
     4201 actcgcgcgc gcagggcgga ggccccaact gcgacttcaa gatatcggcg cccgacctgc
     4261 taaacgcgtt ggtgatggtg cgcagcctgt ggagtgaaaa cttcaccgag ctgccgcgga
     4321 tggttgtgcc tgagtttaag tggagcacta aacaccagta tcgcaacgtg tccctgccag
     4381 tggcgcatag cgatgcgcgg cagaacccct ttgattttta aacggcgcag acggcaagat
     4441 gccatttatc tattttactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4501 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4561 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4621 cagcaagggg gaggattggg aacacaatag caggcatgct ggggatgcgg tgggctctat
     4681 ggccattcct gtagacaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4741 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4801 actcatcaat gtatcttatc atgtctgtga gctgaaggta cgctgtatct cagtcagtca
     4861 agctagccta cttgtacaac tcgtccatac ccagggtaat tccggctgcg gtcacgaact
     4921 caagaagcac catgtgatcc ctcttctcgt tgggatcctt gctcagcttg gactggtagc
     4981 tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt
     5041 agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct
     5101 tgatgccgtt cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact
     5161 ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc
     5221 ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt
     5281 ccttgaagaa gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt
     5341 cgtgctgctt catgtggtcg gggtagcggg cgaagcacat caggccgtag ccgaaggtgg
     5401 tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca
     5461 gcttgccgta ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta
     5521 cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc
     5581 tcaccatggt ggccctaccg gtgaacgttt ttgaacgttt ctaacgtctc tctggatatt
     5641 tcgttctccg cctcgtgggc gtttttaagc tctaaattgt tagaagattc ctcagccaag
     5701 atgacgtctt aattaagaag tctacaggaa tggagaggat tagaggatcg tctcgtgtat
     5761 ggcccccatt atataccctc tagagcgcta tttccctaaa tgtaacagtc acgtcggtta
     5821 tattcaatcc ctaattgtaa cactcacgtg ggttatatac aatccctaaa tgtaacagtc
     5881 acgtcggtta tattcaatcg taccaagact tcgaattcgg atcctgctcg actagagctt
     5941 gcggaaccct tcgaacatgc gtcaatttta cgcagactat ctttctaggg ttaaatcagt
     6001 caggtacata atggatcttt gggtcgccct atagtgagtc gtattacaat tcactggccg
     6061 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     6121 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     6181 aacagttgcg cagcctgaat ggcgaatggc aaattgtaag cgttaatatt ttgttaaaat
     6241 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     6301 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     6361 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     6421 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     6481 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     6541 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     6601 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     6661 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     6721 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     6781 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     6841 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     6901 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     6961 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     7021 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     7081 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     7141 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     7201 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     7261 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     7321 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     7381 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     7441 caggaccact tctgcgctcg tcccttccgg ctggctggtt tattgctgat aaatctggag
     7501 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     7561 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     7621 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     7681 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     7741 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     7801 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     7861 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     7921 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     7981 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     8041 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     8101 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     8161 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     8221 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     8281 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     8341 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     8401 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     8461 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     8521 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     8581 tgagcgagga agcggaagag cgcccaatac
//
