LOCUS       sequence-455846-        8291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Adenoviral helper plasmid containing inducible E2A and L4-22k/33k 
            genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8291)
  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 8291)
  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: JD180
FEATURES             Location/Qualifiers
     source          1..8291
                     /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(404..540)
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     terminator      complement(570..616)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    731..852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      1784..1793
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    join(1991..2009,2010..2032,2033..2051,2052..2074,2075..2093,
                     2094..2116,2117..2135,2136..2158,2159..2177,2178..2200,
                     2201..2219,2220..2242,2243..2261)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     polyA_signal    4139..4363
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4378..4499
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(4549..5262,5263..5265,5266..5268))
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMSEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     4943..4962
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5202..5223
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      5265..5274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     repeat_region   5637..5671
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5708..5727)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5709..5727)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5734..5751)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5734..5750)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5743..5765)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5892..6347)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6029..6050)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6241..6260
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6374..6478
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6479..6547,6548..7339)
                     /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(6697..6716)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7510..8098
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7999..8018
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8252..8269
                     /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 aaagatcgaa atcgagagat cgcctgcagg atagctgacg accttaagga taaatttctg
      421 gtaaggagga cacgtatgga agtgggcaag ttggggaagc cgtatccgtt gctgaatctg
      481 gcatatgtgg gagtataaga cgcgcagcgt cgcatcaggc atttttttct gcgccaatgc
      541 cggtagatag caatccggcc cgaggggcac aaaaaacccc tcaagacccg tttagaggcc
      601 ccaaggggtt atgctacgat accgatagag atgggccaat accacatttg tagaggtttt
      661 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
      721 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
      781 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
      841 caatgtatct tatcatgtct gtgagctgaa ggtacgctgt atctcagtca gtcaagctag
      901 cctagtcctt aagagtcagc gcgcagtatt tactgaagag agcctccgcg tcttccagcg
      961 tgcgccgaag ctgatcttcg cttttgtgat acaggcagct gcgggtgagg gatcgcagag
     1021 acctgttttt tattttcagc tcttgttctt ggcccctgct ctgttgaaat atagcataca
     1081 gagtgggaaa aatcctgttt ctaagctcgc gggtcgatac gggttcgttg ggcgccagac
     1141 gcagcgctcc tcctcctgct gctgccgccg ctgtggattt cttgggcttt gtcagagtct
     1201 tgctatccgg tcgcctttgc ttctgtgtgg ccgctgctgt tgctgccgct gccgccggtg
     1261 cagtatgggc tgtagagatg acggtagtaa tgcaggatgt tacgggggaa ggccacgccg
     1321 tgatggtaga gaagaaagcg gcgggcgaag gagatgttgc ccccacagtc ttgcaagcaa
     1381 gcaactatgg cgttcttgtg cccgcgccat gagcggtagc cttggcgctg ttgttgctct
     1441 tgggctaacg gcggcggctg cttggactta ccggccctgg ttccagtggt gtcccatcta
     1501 cggttgggtc ggcgaacggg cagtgccggc ggcgcctgag gagcggaggt tgtagccatg
     1561 ctggaaccgg ttgccgattt ctggggcgcc ggcgagggga atgcgaccga gggtgacggt
     1621 gtttcgtctg acacctcttc gacctcggaa gcttcctcgt ctaggctctc ccagtcttcc
     1681 atcatgtcct cctcctcctc gtccaaaacc tcctctgcct gactgtccca gtattcctcc
     1741 tcgtccgtgg gtggcggcgg cagctgcagc ttctttttgg gtgccatggt ggccctaccg
     1801 gtgaacgttt ttgaacgttt ctaacgtctc tctggatatt tcgttctccg cctcgtgggc
     1861 gtttttaagc tctaaattgt tagaagattc ctcagccaag attatggagg tcaaaacagc
     1921 gtggatggcg tctccaggcg atctgacggt tcactaaacg agctctgctt atataggtcg
     1981 agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc
     2041 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga
     2101 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc
     2161 cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa
     2221 aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagctcg
     2281 ctggtcgagt atgtcgaggt ggcgtgtacg gtgggaggcc tatataagca gagctcgttt
     2341 agtgaaccgt cagatcgcct ggagaattca gatctcagct ggtcgaccct aggttgtgat
     2401 acaggcagct gcggagcgtt tgccgcgcgt ttctcgtcgc gtccaagacc ctcaaagatt
     2461 tttggcactt cgtcgagcga ggcgatatca ggagaaggaa ggtaccgcca gcatggccag
     2521 tcgggaagag gagcagcgcg aaaccacccc cgagcgcgga cgcggtgcgg cgcgacgtcc
     2581 accaaccatg gaggacgtgt cgtccccgtc gccgtcgccg ccgcctcccc gcgcgccccc
     2641 aaaaaagcgg ctgaggcggc ggctcgagtc cgaggacgaa gaggactcgt cacaagatgc
     2701 gctggtgccg cgcacaccca gcccgcggcc atcgacctcg acggcggatt tggccattgc
     2761 gtccaaaaag aaaaagaagc gcccctctcc caagcccgag cgcccgccat ccccagaggt
     2821 gatcgtggac agcgaggaag aaagagaaga tgtggcgcta caaatggtgg gtttcagcaa
     2881 cccaccggtg ctaatcaagc acggcaaggg aggtaagcgc acggtgcggc ggctgaatga
     2941 ggacgaccca gtggcgcggg gtatgcggac gcaagaggaa aaggaagagt ccagtgaagc
     3001 ggaaagtgaa agcacggtga taaacccgct gagcctgccg atcgtgtctg cgtgggagaa
     3061 gggcatggag gctgcgcgcg cgttgatgga caagtaccac gtggataacg atctaaaggc
     3121 aaacttcaag ctactgcctg accaagtgga agctctggcg gccgtatgca agacctggct
     3181 aaacgaggag caccgcgggt tgcagctgac cttcaccagc aacaagacct ttgtgacgat
     3241 gatggggcga ttcctgcagg cgtacctgca gtcgtttgca gaggtaacct acaagcacca
     3301 cgagcccacg ggctgcgcgt tgtggctgca ccgctgcgct gagatcgaag gcgagcttaa
     3361 gtgtctacac gggagcatta tgataaataa ggagcacgtg attgaaatgg atgtgacgag
     3421 cgaaaacggg cagcgcgcgc tgaaggagca gtctagcaag gccaagatcg tgaagaaccg
     3481 gtggggccga aatgtggtgc agatctccaa caccgacgca aggtgctgcg tgcatgacgc
     3541 ggcctgtccg gccaatcagt tttccggcaa gtcttgcggc atgttcttct ctgaaggcgc
     3601 aaaggctcag gtggctttta agcagatcaa ggctttcatg caggcgctgt atcctaacgc
     3661 ccagaccggg cacggtcacc ttctgatgcc actacggtgc gagtgcaact caaagcctgg
     3721 gcatgcaccc tttttgggaa ggcagctacc aaagttgact ccgttcgccc tgagcaacgc
     3781 ggaggacctg gacgcggatc tgatctccga caagagcgtg ctggccagcg tgcaccaccc
     3841 ggcgctgata gtgttccagt gctgcaaccc tgtgtatcgc aactcgcgcg cgcagggcgg
     3901 aggccccaac tgcgacttca agatatcggc gcccgacctg ctaaacgcgt tggtgatggt
     3961 gcgcagcctg tggagtgaaa acttcaccga gctgccgcgg atggttgtgc ctgagtttaa
     4021 gtggagcact aaacaccagt atcgcaacgt gtccctgcca gtggcgcata gcgatgcgcg
     4081 gcagaacccc tttgattttt aaacggcgca gacggcaaga tgccatttat ctattttact
     4141 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4201 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4261 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4321 gaacacaata gcaggcatgc tggggatgcg gtgggctcta tggccattcc tgtagacaac
     4381 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4441 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4501 catgtctgtg agctgaaggt acgctgtatc tcagtcagtc aagctagcct acttgtacaa
     4561 ctcgtccata cccagggtaa ttccggctgc ggtcacgaac tcaagaagca ccatgtgatc
     4621 cctcttctcg ttgggatcct tgctcagctt ggactggtag ctcaggtagt ggttgtcggg
     4681 cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac
     4741 gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt
     4801 gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat
     4861 gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc
     4921 ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg
     4981 ctcctggacg tagccttcgg acatggcgga cttgaagaag tcgtgctgct tcatgtggtc
     5041 ggggtagcgg gcgaagcaca tcaggccgta gccgaaggtg gtcacgaggg tgggccaggg
     5101 cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc
     5161 gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac
     5221 caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgg tggccctacc
     5281 ggtgaacgtt tttgaacgtt tctaacgtct ctctggatat ttcgttctcc gcctcgtggg
     5341 cgtttttaag ctctaaattg ttagaagatt cctcagccaa gatgacgtct taattaagaa
     5401 gtctacagga atggagagga ttagaggatc gtctcgtgta tggcccccat tatataccct
     5461 ctagagcgct atttccctaa atgtaacagt cacgtcggtt atattcaatc cctaattgta
     5521 acactcacgt gggttatata caatccctaa atgtaacagt cacgtcggtt atattcaatc
     5581 gtaccaagac ttcgaattcg gatcctgctc gactagagct tgcggaaccc ttcgaacatg
     5641 cgtcaatttt acgcagacta tctttctagg gttaaatcag tcaggtacat aatggatctt
     5701 tgggtcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
     5761 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     5821 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     5881 tggcgaatgg caaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     5941 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     6001 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     6061 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     6121 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     6181 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     6241 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     6301 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     6361 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     6421 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     6481 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     6541 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     6601 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     6661 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     6721 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     6781 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     6841 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     6901 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6961 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     7021 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     7081 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     7141 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     7201 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     7261 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     7321 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     7381 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     7441 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     7501 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     7561 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7621 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     7681 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7741 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7801 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7861 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7921 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7981 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     8041 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     8101 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     8161 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     8221 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     8281 gcgcccaata c
//
