LOCUS       sequence-491931-        6895 bp    DNA     circular SYN 05-NOV-2025
DEFINITION  3' AAVLINK plasmid for UNC13A expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6895)
  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  JOURNAL   Unpublished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 6895)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 5, 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            "Unpublished"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'-UNC13A-FLAG
FEATURES             Location/Qualifiers
     source          1..6895
                     /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             3615..3680
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3745..3765)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4158..4298
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4158..4287
                     /label=AAV2 ITR
     rep_origin      4373..4828
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4460..4479)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4670..4691
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4845..4864)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4964..4986
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5024..5042)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5110..5214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5215..5283,5284..6075)
                     /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(5433..5452)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6246..6834
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6735..6754
                     /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 ggtggtaaag gactgtgtga
     1501 aagcctgcct taattctacc tacgagtaca tcttcaataa ctgccatgaa ctgtacagcc
     1561 gggagtacca gacagacccg gccaagaagg gggaagttct cccagaggaa caggggccca
     1621 gcatcaagaa cctcgacttc tggtccaagc tgattaccct catagtgtcc atcattgagg
     1681 aagacaagaa ttcctacact ccctgcctca accagtttcc ccaggagctg aatgtgggta
     1741 aaatcagcgc tgaagtgatg tggaatctgt ttgcccaaga catgaagtac gccatggagg
     1801 agcacgacaa gcatcgtcta tgcaagagtg ccgactacat gaacctccac ttcaaggtga
     1861 aatggctcta caatgagtat gtgacggaac ttcccgcctt caaggaccgc gtgcctgagt
     1921 accctgcatg gtttgaaccc ttcgtcatcc agtggctgga tgagaatgag gaggtgtccc
     1981 gggatttcct gcacggtgcc ctggagcgag acaagaagga tgggttccag cagacctcag
     2041 agcatgccct attctcctgc tccgtggtgg atgtcttctc ccaactcaac cagagctttg
     2101 aaatcatcaa gaaactcgag tgtcccgacc ctcagatcgt ggggcactac atgaggcgct
     2161 ttgccaagac catcagtaat gtgctcctcc agtatgcaga catcatctcc aaggactttg
     2221 cctcctactg ctccaaggag aaggagaaag tgccctgcat tctcatgaat aacactcaac
     2281 agctacgagt tcagctggag aagatgttcg aagccatggg aggaaaggag ctggatgctg
     2341 aagccagtga catcctgaag gagcttcagg tgaaactcaa taacgtcttg gatgagctca
     2401 gccgggtgtt tgctaccagc ttccagccgc acattgaaga gtgtgtcaaa cagatgggtg
     2461 acatccttag ccaggttaag ggcacaggca atgtgccagc cagtgcctgc agcagcgtgg
     2521 cccaggacgc ggacaatgtg ttgcagccca tcatggacct gctggacagc aacctgaccc
     2581 tctttgccaa aatctgtgag aagactgtgc tgaagcgagt gctgaaggag ctgtggaagc
     2641 tggttatgaa caccatggag aaaaccatcg tcctgccgcc cctcactgac cagacgatga
     2701 tcgggaacct cttgagaaaa catggcaagg gattagaaaa gggcagggtg aaattgccaa
     2761 gccactcaga cggaacccag atgatcttca atgcagccaa ggagctgggt cagctgtcca
     2821 aactcaagga tcacatggta cgagaagaag ccaagagctt gaccccaaag cagtgcgcgg
     2881 ttgttgagtt ggccctggac accatcaagc aatatttcca cgcgggtggc gtgggcctca
     2941 agaagacctt cctggagaag agcccggacc tgcaatcctt gcgctatgcc ctgtcgctct
     3001 acacgcaggc caccgacctg ctaatcaaga cctttgtaca gacgcaatcg gcccagggct
     3061 tgggtgtaga agaccctgtg ggtgaagtct ctgtccatgt tgagctgttc actcatccag
     3121 gaactgggga acacaaggtc acagtgaaag tggtggctgc caatgacctc aagtggcaga
     3181 cttctggcat cttccggccg ttcatcgagg tcaacatcat tgggccccag ctcagcgaca
     3241 agaaacgcaa gtttgcgacc aaatccaaga acaatagctg ggctcccaag tacaatgaga
     3301 gcttccagtt cacgctgagc gccgacgcgg gtcccgagtg ctatgagctg caggtgtgcg
     3361 tcaaggacta ctgcttcgcg cgcgaggacc gcacggtggg gctggccgtg ctgcagctgc
     3421 gtgagctggc ccagcgcggg agcgccgcct gctggctgcc gctcggccgc cgcatccaca
     3481 tggacgacac gggcctcacg gtgctgcgaa tcctctcgca gcgcagcaac gacgaggtgg
     3541 ccaaggagtt cgtgaagctc aagtcggaca cgcgctccgc cgaggagggc ggtgccgcgc
     3601 ctgcgccttg tacagactac aaagaccatg acggtgatta taaagatcat gacatcgact
     3661 acaaggatga cgatgacaag taactcgaga taatcaacct ctggattaca aaatttgtga
     3721 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     3781 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     3841 atcctggtta gttcttgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     3901 aggggctcgg ctgttgggca ctgacaattc cgtggtgttt atttgtgaaa tttgtgatgc
     3961 tattgcttta tttgtaacca tctagcttta tttgtgaaat ttgtgatgct attgctttat
     4021 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     4081 ttcaggttca gggggagatg tgggaggttt tttaaagatc tggagaccgg tgtggcacgt
     4141 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4201 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4261 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4321 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4381 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4441 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4501 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4561 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4621 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4681 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     4741 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4801 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4861 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4921 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4981 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5041 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5101 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5161 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5221 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5281 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5341 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5401 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5461 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5521 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5581 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5641 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5701 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5761 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5821 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5881 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5941 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6001 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6061 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6121 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6181 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6241 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6301 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6361 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6421 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6481 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6541 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6601 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6661 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6721 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6781 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6841 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
