LOCUS       sequence-491884-        6459 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  5' AAVLINK plasmid for TOP2B expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6459)
  AUTHORS   Zhonghua Lu Lab
  TITLE     AAVLINK: A Potent DNA-Recombination Method for Large Cargo Delivery 
            in Gene Therapy
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6459)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAVLINK-EF1alpha-HA-5'-TOP2B
FEATURES             Location/Qualifiers
     source          1..6459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        175..386
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             399..667
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             704..730
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     RBS             3267..3275
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     repeat_region   3733..3862
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      3937..4392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4024..4043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4234..4255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4409..4428)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4528..4550
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4588..4606)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4674..4778
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4779..4847,4848..5639)
                     /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(4997..5016)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5810..6398
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6299..6318
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtggatct gcgatcgctc cggtgcccgt cagtgggcag
      181 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaacgggtg
      241 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
      301 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
      361 gcaacgggtt tgccgccaga acacagctga agcttcgagg ggctcgcatc tctccttcac
      421 gcgcccgccg ccctacctga ggccgccatc cacgccggtt gagtcgcgtt ctgccgcctc
      481 ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt aagtttaaag ctcaggtcga
      541 gaccgggcct ttgtccggcg ctcccttgga gcctacctag actcagccgg ctctccacgc
      601 tttgcctgac cctgcttgct caactctacg tctttgtttc gttttctgtt ctgcgccgtt
      661 acagatccaa gctgtgaccg gcgcctaccg tacggccacc atgtaccctt atgacgtacc
      721 tgactatgct ggatctgcca agtcgggtgg ctgcggcgcg ggagccggcg tgggcggcgg
      781 caacggggca ctgacctggg tgactctttt tgatcagaac aatgctgcaa aaaaagaaga
      841 gtcagaaact gccaacaaaa atgattcttc aaagaagttg tctgttgaga gagtgtatca
      901 gaagaagaca caacttgaac acattcttct tcgtcctgat acatatattg ggtcagtgga
      961 gccattgacg cagttcatgt gggtgtatga tgaagatgta ggaatgaatt gcagggaggt
     1021 tacctttgtg ccaggtttat acaagatctt tgatgaaatt ttggttaatg ctgctgacaa
     1081 taaacagagg gataagaaca tgacttgtat taaagtttct attgatcctg aatctaacat
     1141 tataagcatt tggaataatg ggaaaggcat tccagtagta gaacacaagg tagagaaagt
     1201 ttatgttcct gctttaattt ttggacagct tttaacatcc agtaactatg atgatgatga
     1261 gaaaaaagtt acaggtggtc gtaatggtta tggtgcaaaa ctttgtaata ttttcagtac
     1321 aaagtttaca gtagaaacag cttgcaaaga atacaaacac agttttaagc agacatggat
     1381 gaataatatg atgaagactt ctgaagccaa aattaaacat tttgatggtg aagattacac
     1441 atgcataaca ttccaaccag atctgtccaa atttaagatg gaaaaacttg acaaggatat
     1501 tgtggccctc atgactagaa gggcatatga tttggctggt tcgtgtagag gggtcaaggt
     1561 catgtttaat ggaaagaaat tgcctgtaaa tggatttcgc agttatgtag atctttatgt
     1621 gaaagacaaa ttggatgaaa ctggggtggc cctgaaagtt attcatgagc ttgcaaatga
     1681 aagatgggat gtttgtctca cattgagtga aaaaggattc cagcaaatca gctttgtaaa
     1741 tagtattgca actacaaaag gtggacggca cgtggattat gtggtagatc aagttgttgg
     1801 taaactgatt gaagtagtta agaaaaagaa caaagctggt gtatcagtga aaccatttca
     1861 agtaaaaaac catatatggg tttttattaa ttgccttatt gaaaatccaa cttttgattc
     1921 tcagactaag gaaaacatga ctctgcagcc caaaagtttt gggtctaaat gccagctgtc
     1981 agaaaaattt tttaaagcag cctctaattg tggcattgta gaaagtatcc tgaactgggt
     2041 gaaatttaag gctcagactc agctgaataa gaagtgttca tcagtaaaat acagtaaaat
     2101 caaaggtatt cccaaactgg atgatgctaa tgatgctggt ggtaaacatt ccctggagtg
     2161 tacactgata ttaacagagg gagactctgc caaatcactg gctgtgtctg gattaggtgt
     2221 gattggacga gacagatacg gagtttttcc actcaggggc aaaattctta atgtacggga
     2281 agcttctcat aaacagatca tggaaaatgc tgaaataaat aatattatta aaatagttgg
     2341 tctacaatat aagaaaagtt acgatgatgc agaatctctg aaaaccttac gctatggaaa
     2401 gattatgatt atgaccgatc aggatcaaga tggttctcac ataaaaggcc tgcttattaa
     2461 tttcatccat cacaattggc catcactttt gaagcatggt tttcttgaag agttcattac
     2521 tcctattgta aaggcaagca aaaataagca ggaactttcc ttctacagta ttcctgaatt
     2581 tgacgaatgg aaaaaacata tagaaaacca gaaagcctgg aaaataaagt actataaagg
     2641 attgggtact agtacagcta aagaagcaaa ggaatatttt gctgatatgg aaaggcatcg
     2701 catcttgttt agatatgctg gtcctgaaga tgatgctgcc attaccttgg catttagtaa
     2761 gaagaagatt gatgacagaa aagaatggtt aacaaatttt atggaagacc ggagacagcg
     2821 taggctacat ggcttaccag agcaattttt atatggtact gcaacaaagc atttgactta
     2881 taatgatttc atcaacaagg aattgattct cttctcaaac tcagacaatg aaagatctat
     2941 accatctctt gttgatggct ttaaacctgg ccagcggaaa gttttattta cctgtttcaa
     3001 gaggaatgat aaacgtgaag taaaagttgc ccagttggct ggctctgttg ctgagatgtc
     3061 ggcttatcat catggagaac aagcattgat gatgactatt gtgaatttgg ctcagaactt
     3121 tgtgggaagt aacaacatta acttgcttca gcctattggt cagtttggaa ctcggcttca
     3181 tggtggcaaa gatgctgcaa gccctcgtta tattttcaca atgttaagca ctttagcaag
     3241 gctacttttt cctgctgtgg atgataacct ccttaagttc ctttatgatg ataatcaacg
     3301 tgtagagcct gagtggtata ttcctataat tcccatggtt ttaataaatg gtgctgaggg
     3361 cattggtact ggatgggctt gtaaactacc caactatgat gctagggaaa ttgtgaacaa
     3421 tgtcagacga atgctagatg gcctggatcc tcatcccatg cttccaaact acaaaaactt
     3481 taaaggcacg attcaagaac ttggtcaaaa ccagtatgca gtcagtggtg aaatatttgt
     3541 agtggacaga aacacagtag aaattacaga gcttccagtt agaacttgga cacaggtata
     3601 taaagaacag gtaagtatca aggttacaag acaggtttaa ggagaccaat aagctattat
     3661 tcgtatagga tactttatac gaagttatta agttatggag accggtgtgg cacgtgcgga
     3721 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     3781 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggcggcctc agtgagcgag
     3841 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3901 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3961 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     4021 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4081 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4141 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4201 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4261 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     4321 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4381 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4441 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4501 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4561 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4621 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4681 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4741 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4801 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4861 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4921 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4981 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5041 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5101 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5161 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5221 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5281 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5341 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5401 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5461 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     5521 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5581 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5641 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5701 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5761 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5821 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5881 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5941 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6001 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6061 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6121 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6181 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6241 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6301 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6361 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6421 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
