LOCUS       sequence-476867-        6454 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for ITSN1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6454)
  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  JOURNAL   Unpublished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 6454)
  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            "Unpublished"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' ITSN1
FEATURES             Location/Qualifiers
     source          1..6454
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     721..740
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             2431..2496
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2561..2581)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2974..3114
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2974..3103
                     /label=AAV2 ITR
     rep_origin      3189..3644
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3276..3295)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3486..3507
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3661..3680)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3780..3802
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3840..3858)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3926..4030
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4031..4099,4100..4891)
                     /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(4249..4268)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5062..5650
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5551..5570
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5712..5841
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             join(5988..6454,1..565)
                     /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"
ORIGIN
        1 ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac gtatagccga
       61 aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa tgttaatcca
      121 tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac ttagcctggg
      181 ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg atccgaataa
      241 ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct
      301 atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt acggcgctaa
      361 ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg tcggagccgc
      421 gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg gtggctggac
      481 caatgtaaat attgtaatga actatatccg taacctggat agtgaaacag gggcaatggt
      541 gcgcctgctg gaagatggcg attaggtaag tatcaaggtt acaagacagg tttaaggaga
      601 ccaataacta gtataacttc gtataggata ctttatagca atttatgaaa ctgggcttgt
      661 cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact
      721 ttgcctttct ctccacaggt gattgggatg tacgactaca ccgcgcagaa tgacgatgag
      781 ctggccttca acaagggcca gatcatcaac gtcctcaaca aggaggaccc tgactggtgg
      841 aaaggagaag tcaatggaca agtggggctc ttcccatcca attatgtgaa gctgaccaca
      901 gacatggacc caagccagca atggtgttca gacttacatc tcttggatat gttgacccca
      961 actgaaagaa agcgacaagg atacatccac gagctcattg tcaccgagga gaactatgtg
     1021 aatgacctgc agctggtcac agagattttt caaaaacccc tgatggagtc tgagctgctg
     1081 acagaaaaag aggttgctat gatttttgtg aactggaagg agctgattat gtgtaatatc
     1141 aaactactaa aagcgctgag agtccgcaag aagatgtccg gggagaagat gcctgtgaag
     1201 atgattggag acatcctgag cgcacagctg ccgcacatgc agccctacat ccgcttctgc
     1261 agccgccagc tcaacggggc tgccctgatc cagcagaaga cggacgaggc cccagacttc
     1321 aaggagttcg tcaaaagatt ggcaatggat cctcggtgta aagggatgcc actctctagt
     1381 tttatactga agcctatgca acgggtaaca agatacccac tgatcattaa aaatatcctg
     1441 gaaaacaccc ctgaaaacca cccggaccac agccacttga agcacgccct ggagaaggcg
     1501 gaagagctct gttcccaggt gaacgaaggg gtgcgggaga aggagaactc tgaccggctg
     1561 gagtggatcc aggcccacgt gcagtgtgaa ggcctgtctg agcaacttgt gttcaattca
     1621 gtgaccaatt gcttggggcc gcgcaaattt ctgcacagtg ggaagctcta caaggccaag
     1681 agcaacaagg agctgtatgg cttccttttc aacgacttcc tcctgctgac tcagatcacg
     1741 aagcctttgg ggtcttctgg caccgacaaa gtcttcagcc ccaaatcaaa cctgcagtat
     1801 aaaatgtata aaacacctat tttcctaaat gaggttctag taaaattacc caccgaccct
     1861 tctggagacg agcccatctt ccacatctcc cacattgacc gcgtctatac tctccgagca
     1921 gaaagcataa atgaaaggac tgcctgggtg cagaaaatca aagctgcttc tgaactctac
     1981 atagagactg agaaaaagaa gcgcgagaaa gcgtacctgg tccgttccca aagggcaaca
     2041 ggcattggaa ggttgatggt gaacgtggtt gaaggcatcg agttgaaacc ctgtcggtca
     2101 catggaaaga gcaacccgta ctgtgaggtg accatgggtt cccagtgcca catcaccaag
     2161 acgatccagg acactctgaa ccccaagtgg aattccaact gccagttctt catccgagac
     2221 ctggagcagg aagtcctctg catcactgtg ttcgagaggg accagttctc accagatgat
     2281 tttttgggtc ggacggagat ccgcgtggcg gacatcaaga aagaccaggg ctccaaaggt
     2341 ccagttacga agtgtcttct gctgcacgaa gtccccacgg gagagattgt ggtccgcttg
     2401 gacctgcagt tgtttgatga gccgtgtaca gactacaaag accatgacgg tgattataaa
     2461 gatcatgaca tcgactacaa ggatgacgat gacaagtaac tcgagataat caacctctgg
     2521 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     2581 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     2641 tctcctcctt gtataaatcc tggttagttc ttgccacggc ggaactcatc gccgcctgcc
     2701 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgtttattt
     2761 gtgaaatttg tgatgctatt gctttatttg taaccatcta gctttatttg tgaaatttgt
     2821 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     2881 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagatctgga
     2941 gaccggtgtg gcacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     3001 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     3061 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     3121 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     3181 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3241 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     3301 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3361 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     3421 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3481 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     3541 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     3601 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     3661 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3721 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3781 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     3841 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3901 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3961 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4021 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4081 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4141 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4201 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4261 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4321 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4381 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4441 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4501 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4561 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4621 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4681 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4741 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4801 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4861 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4921 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4981 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5041 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5101 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5161 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5221 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5281 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5341 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5401 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5461 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5521 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5581 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5641 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5701 tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc
     5761 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
     5821 actccatcac taggggttcc tgcggccgca cgcgtcgctc tgcgcgaggc ggctccgccg
     5881 cggcggaggg atacggcgca ccatatatat atcgcggggc gcagactcgc gctccggcag
     5941 tggtgctggg agtgtcgtgg acgccgtgcc gttactcgta cgccaccatg tccaatttac
     6001 tgaccgtaca ccaaaatttg cctgcattac cggtcgatgc aacgagtgat gaggttcgca
     6061 agaacctgat ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc
     6121 ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc
     6181 ccgcagaacc tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag
     6241 taaaaaccat ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tccgggctgc
     6301 cacgaccaag tgacagcaat gctgtttcac tggttatgcg gcggatccga aaagaaaacg
     6361 ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg
     6421 ttcgttcact catggaaaat agcgatcgct gcca
//
