LOCUS       sequence-476870-        6772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for UBN2 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6772)
  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  JOURNAL   Unpublished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 6772)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAVLINKv1-pCALM1-Cre-3' UBN2
FEATURES             Location/Qualifiers
     source          1..6772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1921..1940)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             complement(2096..3127)
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     repeat_region   3274..3403
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(3465..4053)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3545..3564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4224..5015,5016..5084))
                     /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     4847..4866
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5085..5189)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5257..5275
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5313..5335)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5435..5454
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5471..5926)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5608..5629)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5820..5839
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6001..6141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6012..6141
                     /label=AAV2 ITR
     primer_bind     6534..6554
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6619..6684)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
ORIGIN
        1 gagtgggaaa gcgttgctgc atgctgagct cctcctggct gtaaaccctt tagtaaattc
       61 tgagttaggc tatggtggaa agcggctgag gtagatcccg aggtggttgt cactccagct
      121 gtgtccgttc caaagccaaa tatctccaag ggaaactgga agggcatggt cactggaaca
      181 aggccaccca gcatcccaaa aggagtcaca ttttgatttg ttacagacaa aggtgatgtc
      241 atgagagtca gaggtgaggc attagccaat aatgatgctc ctgctgtgga ctgcacactg
      301 gctgttacca ctgaagaccc tgaggcagtg gatggtctat gtggagtaga caatggtttg
      361 gtagcaccct gtgttccact tcctgttccc ccagacagac tagtcctatt agctccgctt
      421 gagttaaggt tgctacctct gctggcaggt acattcattg caattccact caagctgttc
      481 ttcccaacag ttccagtgga tgatgagttt gttagctttg agggggcctg aagatttttt
      541 gtaggtggaa ggcctgaagt gcgactagag ggcaataaat tcaaggtggg agactgtctg
      601 ctgatgttca ttccactggg ctgtttatgg actgggctgt tagtgctgga gtggctaccc
      661 tgggcaacta gtgcatttgg actggaacta cttggggata cagtaggctt gggagttggg
      721 ttggatttag aaataagttt agttgatact gaaggcttag ctgacagaga gggcttgggt
      781 gaggcagaag gcttaggcga gggcaggggt ttaggagatg tggcaggctt gggagatgcg
      841 gcaagttttg gggaggcagc catcgagaag ggagatttga aaccctgcgt ggagatctgt
      901 gacaccatag ccttggctaa atagttactg gaggtagtgg tgctaggtac tgtcctagaa
      961 accagggggt gggacccttg agaaccattt ccaggtgagg gggtagataa gggaaggcgg
     1021 tacataagtg gcttatctga agtcttaata agtgaggagg aacaggagag ttggggatta
     1081 ttacttaact tcacgacggg gttggtctgg gatttactga tggtggcctg taatggagac
     1141 acatagttct gctggacagc tgaatgctgg tgcacctttg ttacttgtgt taacgaagaa
     1201 gcatcttggg cctcggatgt tcccagagca tgagaagagg cagcaatttg ggcttgggaa
     1261 gaagaggaga catgggtctg tgaagaggaa gaagtgtgaa tctggcttga cctctgaagt
     1321 ccttgttgaa gtagcctggc tggcaaaggt tctaaagaaa ctttagggtt ctgaatggaa
     1381 gaaccagcaa taaggcctga agagatgcca gtatgagcta aatcctgggg tttctttggt
     1441 actggccctg tgtgaccagc aataagactt gaagaatgtg tagtctgagt agaatctagt
     1501 tttttgggag tagctagtgg cagcttactc atgatacttg ctaatttttc ttctctcagt
     1561 cctggacgag gctttgtggt tggcatgctt atccttgagc caactggagg gcccttgttc
     1621 ccattgttga taaccgctaa agcttcagaa acaagatcca gtgaaggtga atggaaagaa
     1681 aggtcttcat ctagtgagtc gtcgaggcag atggtttctt gggctggtgc agagctagag
     1741 ctagctgcag caatggcagc tgtgctggag ctcgttggag gaccgctaac cgaagccacc
     1801 agggatgttg gagttttctg gtcctttttt ggactacact ccttaagcat agtggggaaa
     1861 gaatggagag gaagggtctt taccatcacc tcctttactt tgggtttagg tgcaggaatc
     1921 acctgtggag agaaaggcaa agtggatgtc agtaagacca ataggtgcct atcagaaacg
     1981 caagagtctt ctctgtctcg acaagcccag tttcataaat tgctataaag tatcctatac
     2041 gaagttatac tagttattgg tctccttaaa cctgtcttgt aaccttgata cttacctaat
     2101 cgccatcttc cagcaggcgc accattgccc ctgtttcact atccaggtta cggatatagt
     2161 tcattacaat atttacattg gtccagccac cagcttgcat gatctccggt attgaaactc
     2221 cagcgcgggc catatctcgc gcggctccga cacgggcact gtgtccagac caggccaggt
     2281 atctctgacc agagtcatcc ttagcgccgt aaatcaatcg atgagttgct tcaaaaatcc
     2341 cttccagggc gcgagttgat agctggctgg tggcagatgg cgcggcaaca ccattttttc
     2401 tgacccggca aaacaggtag ttattcggat catcagctac accagagacg gaaatccatc
     2461 gctcgaccag tttagttacc cccaggctaa gtgccttctc tacacctgcg gtgctaacca
     2521 gcgttttcgt tctgccaata tggattaaca ttctcccacc gtcagtacgt gagatatctt
     2581 taaccctgat cctggcaatt tcggctatac gtaacagggt gttataagca atccccagaa
     2641 atgccagatt acgtatatcc tggcagcgat cgctattttc catgagtgaa cgaacctggt
     2701 cgaaatcagt gcgttcgaac gctagagcct gttttgcacg ttcaccggca tcaacgtttt
     2761 cttttcggat ccgccgcata accagtgaaa cagcattgct gtcacttggt cgtggcagcc
     2821 cggaccgacg atgaagcatg tttagctggc ccaaatgttg ctggatggtt tttactgcca
     2881 gaccgcgcgc ctgaagatat agaagataat cgcgaacatc ttcaggttct gcgggaaacc
     2941 atttccggtt attcaacttg caccatgccg cccacgaccg gcaaacggac agaagcattt
     3001 tccaggtatg ctcagaaaac gcctggcgat ccctgaacat gtccatcagg ttcttgcgaa
     3061 cctcatcact cgttgcatcg accggtaatg caggcaaatt ttggtgtacg gtcagtaaat
     3121 tggacatggt ggcgtacgag taacggcacg gcgtccacga cactcccagc accactgccg
     3181 gagcgcgagt ctgcgccccg cgatatatat atggtgcgcc gtatccctcc gccgcggcgg
     3241 agccgcctcg cgcagagcga cgcgtgcggc cgcaggaacc cctagtgatg gagttggcca
     3301 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     3361 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggacatgtg agcaaaaggc
     3421 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3481 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3541 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3601 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3661 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3721 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3781 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3841 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3901 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3961 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4021 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4081 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4141 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4201 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4261 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4321 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4381 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4441 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4501 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4561 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4621 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4681 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4741 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4801 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4861 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4921 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4981 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5041 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5101 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5161 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     5221 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     5281 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     5341 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     5401 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     5461 gtgcaccata aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     5521 atcagctcat tttttaacca atagaccgaa atcggcaaaa tcccttataa atcaaaagaa
     5581 tagcccgaga tagagttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     5641 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     5701 ccatcaccca aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     5761 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     5821 gggaagaaag cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt cacgctgcgc
     5881 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt
     5941 gacgtatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
     6001 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
     6061 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
     6121 actccatcac taggggttcc tgcggccgct cggtccgcac gtgccacacc ggtctccaga
     6181 tctttaaaaa acctcccaca tctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     6241 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     6301 ttcacaaata aagctagatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     6361 acaccacgga attgtcagtg cccaacagcc gagcccctgt ccagcagcgg gcaaggcagg
     6421 cggcgatgag ttccgccgtg gcaagaacta accaggattt atacaaggag gagaaaatga
     6481 aagccatacg ggaagcaata gcatgataca aaggcattaa agcagcgtat ccacatagcg
     6541 taaaaggagc aacatagtta agaataccag tcaatctttc acaaattttg taatccagag
     6601 gttgattatc tcgagttact tgtcatcgtc atccttgtag tcgatgtcat gatctttata
     6661 atcaccgtca tggtctttgt agtctgtaca ctgagatttc cgtggtaatt tagtgtcccc
     6721 tttactttgg cctccatcgt gaaatgcttg ctgtaagtgt gcaggcagag gt
//
