LOCUS       sequence-491396-        6289 bp    DNA     circular SYN 03-NOV-2025
DEFINITION  3' AAVLINK plasmid for MBD5 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6289)
  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  JOURNAL   Unpublished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 6289)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 3, 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            "Unpublished"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'-MBD5-FLAG
FEATURES             Location/Qualifiers
     source          1..6289
                     /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"
     CDS             3009..3074
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3139..3159)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3552..3692
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3552..3681
                     /label=AAV2 ITR
     rep_origin      3767..4222
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3854..3873)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4064..4085
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4239..4258)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4358..4380
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4418..4436)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4504..4608
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4609..4677,4678..5469)
                     /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(4827..4846)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5640..6228
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6129..6148
                     /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 ggggtaccag aatctccagg
     1501 cgttccaagg acagtccaca attccttgcc cagctaacaa taaccccatg gcttgtctgt
     1561 ttcagaactt tcaggtgaga atgcaggaag atgcagctct cctaaacaaa agaataagca
     1621 ctcagcctgg gctcacagca cttcctgaga atccaaacac tacacttcca ccttttcaag
     1681 atacaccttg tgagttgcaa ccgaggattg acccatctct tggtcaacag gtgaaggatg
     1741 gcctcgttgt gggtggccca ggtgatgctt ccgtagatgc catttacaaa gcagttgtcg
     1801 atgcagccag caaaggaatg caggttgtca tcaccactgc agtcaacagt acaactcaga
     1861 tcagccccat tccagctctg agtgccatga gtgccttcac tgcctcaatt ggtgacccat
     1921 taaatctctc cagtgttgtc agtgcggtca ttcatggacg gaacatggga ggtgttgatc
     1981 atgatggtag gctgaggaat tcaagagggg ctcggctgcc caagaatcta gaccatggga
     2041 aaaatgtgaa cgaaggagat gggtttgaat atttcaagtc agcaagttgc cacacatcca
     2101 aaaaacagtg ggacggggag caaagcccca gaggggagcg aaacaggtgg aagtacgagg
     2161 aatttttaga tcatccaggc catatccaca gtagtccttg tcatgaaagg cccaacaatg
     2221 tctctacact gccatttctg cctggggaac agcacccaat actgttacca ccaagaaact
     2281 gtccagggga taaaattcta gaggaaaatt tcaggtataa taactacaaa agaactatga
     2341 tgagttttaa ggagagacta gagaacactg tggaaagatg tgcacacata aatgggaata
     2401 gacctcgaca gagtcgggga tttggagagc tgctaagcac tgcaaagcaa gacctggtcc
     2461 tagaggagca gtctccaagt tcctcaaata gtttggaaaa ttctctggtc aaagactaca
     2521 tccattacaa tggagacttt aatgccaaaa gcgttaatgg gtgtgtgcct agcccttcag
     2581 atgctaaaag cattagtagt gaagatgacc taaggaaccc agactccccc tcttcaaatg
     2641 aattgataca ttatagacca aggacgttca atgttggcga cttggtctgg ggccaaatca
     2701 aaggactgac ttcctggcct ggaaaattag taagagaaga cgacgttcac aattcatgtc
     2761 aacaaagccc cgaggaaggg aaggtggagc ccgagaagtt gaagacacta acagaaggtt
     2821 tggaagccta cagccgtgtc cggaaaagga acagaaaaag tggaaagcta aataaccatt
     2881 tagaagctgc tattcatgag gccatgagtg aactggacaa aatgtctggg actgtacacc
     2941 aaatcccaca gggtgacaga caaatgagac cccccaaacc caagaggagg aagatctcca
     3001 gatgtacaga ctacaaagac catgacggtg attataaaga tcatgacatc gactacaagg
     3061 atgacgatga caagtaactc gagataatca acctctggat tacaaaattt gtgaaagatt
     3121 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3181 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3241 gttagttctt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3301 tcggctgttg ggcactgaca attccgtggt gtttatttgt gaaatttgtg atgctattgc
     3361 tttatttgta accatctagc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     3421 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     3481 ttcaggggga gatgtgggag gttttttaaa gatctggaga ccggtgtggc acgtgcggac
     3541 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3601 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     3661 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     3721 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     3781 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3841 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3901 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3961 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     4021 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4081 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     4141 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4201 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     4261 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4321 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4381 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4441 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     4501 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4561 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4621 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4681 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4741 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4801 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4861 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4921 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     4981 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5041 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5101 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5161 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5221 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5281 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5341 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5401 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5461 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5521 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5581 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5641 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5701 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5761 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5821 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5881 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5941 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6001 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6061 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6121 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6181 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6241 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
