LOCUS       sequence-491794-        6541 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  3' AAVLINK plasmid for ERBIN expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6541)
  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  JOURNAL   Unpublished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 6541)
  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            "Unpublished"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: pAAVLINKv1-pCALM1-Cre-3'-ERBIN-FLAG
FEATURES             Location/Qualifiers
     source          1..6541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             277..1308
                     /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"
     primer_bind     1464..1483
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             complement(2453..2464)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3261..3326
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3391..3411)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3804..3944
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3804..3933
                     /label=AAV2 ITR
     rep_origin      4019..4474
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4106..4125)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4316..4337
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4491..4510)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4610..4632
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4670..4688)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4756..4860
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4861..4929,4930..5721)
                     /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(5079..5098)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5892..6480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6381..6400
                     /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 gcgtcgctct gcgcgaggcg gctccgccgc ggcggaggga
      181 tacggcgcac catatatata tcgcggggcg cagactcgcg ctccggcagt ggtgctggga
      241 gtgtcgtgga cgccgtgccg ttactcgtac gccaccatgt ccaatttact gaccgtacac
      301 caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg
      361 gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt
      421 tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct
      481 gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaaccatc
      541 cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt
      601 gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt
      661 gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc
      721 atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat
      781 aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact
      841 gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt
      901 gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct
      961 ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc
     1021 gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact
     1081 catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga
     1141 cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag
     1201 atcatgcaag ctggtggctg gaccaatgta aatattgtaa tgaactatat ccgtaacctg
     1261 gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattaggt aagtatcaag
     1321 gttacaagac aggtttaagg agaccaataa ctagtataac ttcgtatagg atactttata
     1381 gcaatttatg aaactgggct tgtcgagaca gagaagactc ttgcgtttct gataggcacc
     1441 tattggtctt actgacatcc actttgcctt tctctccaca ggtaaatgga cattctgagg
     1501 aaacttccca gtctcctaat aggactgaac cacatgacag tgattgttct gttgacttag
     1561 gtatttccaa aagcactgaa gatctctccc ctcagaaaag tggtccagtt ggatctgttg
     1621 tgaaatctca tagcataact aatatggaga ttggagggct aaaaatctat gatattctta
     1681 gtgataatgg acctcagcag ccaagtacaa ccgttaaaat cacatctgct gttgatggaa
     1741 aaaatatagt caggagcaag tctgccacac tgttgtatga tcaaccattg caggtattta
     1801 ctggttcttc ctcatcttct gatttaatat caggaacaaa ggcaattttc aagtttgatt
     1861 caaatcataa tcccgaagag ccaaatataa taagaggccc cacaagtggc ccacaatctg
     1921 cacctcaaat atatggtcct ccacagtata atatccaata cagtagcagt gctgcagtca
     1981 aagacacttt gtggcactcc aaacaaaatc cccaaataga ccatgccagt tttcctcctc
     2041 agctccttcc tagatcagag agcacagaaa atcaaagtta tgctaaacat tctgccaata
     2101 tgaatttctc taatcataac aatgttcgag ctaatactgc ataccattta catcagagac
     2161 ttggcccagc aagacatggg gaaatgtggg ccatctcacc aaacgaccga cttattcctg
     2221 cagtaactcg aagtacaatc cagcgacaaa gtagtgtgtc ctccacagcc tctgtaaatc
     2281 ttggtgatcc aggctctaca aggcgggctc agattcctga aggagattat ttatcataca
     2341 gagagttcca ctcagcggga agaactcctc caatgatgcc aggatcacag agaccccttt
     2401 ctgcacgaac atacagcata gatggtccaa atgcatcaag acctcagagt gctcgaccct
     2461 ctattaatga aataccagag agaactatgt cagttagtga tttcaattat tcacggacta
     2521 gtccttcaaa aagaccaaat gcaagggttg gttctgagca ttctttatta gatcctccag
     2581 gaaaaagtaa agttcctcgt gactggagag aacaagtact tcgacatatt gaagccaaaa
     2641 agttagaaaa gaagcatccc cagacatcca gttcaggaga tccttgtcaa gatggtatat
     2701 tcatttcagg acagcagaac tactcatcag ccacacttag tcacaaagat gttcctccag
     2761 acagcttgat gaaaatgcct ttgagtaatg gacagatggg ccagcctctc aggcctcagg
     2821 caaattatag tcaaatacat cacccccctc aggcatctgt ggcaaggcat ccctctagag
     2881 aacaactaat tgattacttg atgctgaaag tggcccacca gcctccatat acacagcccc
     2941 attgttctcc tagacaaggc catgaactgg caaaacaaga gattcgagtg agggttgaaa
     3001 aggatccaga acttggattt agcatatcag gtggtgtcgg gggtagagga aacccattca
     3061 gacctgatga tgatggtata tttgtaacaa gggtacaacc tgaaggacca gcatcaaaat
     3121 tactgcagcc aggtgataaa attattcagg ctaatggcta cagttttata aatattgaac
     3181 atggacaagc agtgtccttg ctaaaaactt tccagaatac agttgaactc atcattgtac
     3241 gagaagtttc ctcatgtaca gactacaaag accatgacgg tgattataaa gatcatgaca
     3301 tcgactacaa ggatgacgat gacaagtaac tcgagataat caacctctgg attacaaaat
     3361 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3421 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3481 gtataaatcc tggttagttc ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3541 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgtttattt gtgaaatttg
     3601 tgatgctatt gctttatttg taaccatcta gctttatttg tgaaatttgt gatgctattg
     3661 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     3721 ttatgtttca ggttcagggg gagatgtggg aggtttttta aagatctgga gaccggtgtg
     3781 gcacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     3841 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     3901 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     3961 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     4021 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4081 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4141 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4201 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     4261 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4321 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     4381 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4441 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     4501 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4561 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4621 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     4681 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     4741 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4801 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4861 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4921 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4981 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5041 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5101 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5161 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5221 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5281 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5341 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5401 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5461 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5521 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5581 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5641 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5701 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5761 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5821 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5881 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5941 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6001 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6061 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6121 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6181 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6241 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6301 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6361 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6421 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6481 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6541 t
//
