LOCUS       sequence-491432-        7168 bp    DNA     circular SYN 03-NOV-2025
DEFINITION  3' AAVLINK plasmid for SHANK2 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7168)
  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  JOURNAL   Unpublished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 7168)
  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            "Unpublished"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'-SHANK2-FLAG
FEATURES             Location/Qualifiers
     source          1..7168
                     /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             3888..3953
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4018..4038)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4431..4571
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4431..4560
                     /label=AAV2 ITR
     rep_origin      4646..5101
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4733..4752)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4943..4964
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5118..5137)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5237..5259
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5297..5315)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5383..5487
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5488..5556,5557..6348)
                     /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(5706..5725)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6519..7107
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7008..7027
                     /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 gggcagcagc atggagatcg
     1501 acccccaggc cccggagcca ccgagccagc tgcggcctga cgaaagcctg accgtcagca
     1561 gcccctttgc cgccgccatc gccggagccg tccgcgaccg tgagaagcgg ctggaagcca
     1621 ggaggaactc cccggccttc ctctccacag acctggggga tgaggatgtg ggcctggggc
     1681 cacccgcccc caggacgcgg ccctccatgt tccccgagga gggggatttt gctgacgagg
     1741 acagcgctga gcagctgtca tcccccatgc cgagtgccac gcccagggag cccgaaaacc
     1801 atttcgtggg tggcgccgag gccagtgctc cgggtgaggc tgggaggccg ctgaattcca
     1861 cgtccaaagc ccaggggccc gagagcagcc cagcagtgcc ctccgcgagc agcggcacag
     1921 ccggccccgg gaattatgtc cacccactca cagggcggct gcttgatccc agctccccgc
     1981 tggccctggc actctccgca agggaccgag ccatgaagga gtctcaacag ggacccaaag
     2041 gggaggcccc caaggccgac ctcaacaaac ctctttacat tgataccaaa atgcggccca
     2101 gcctggatgc cggcttccct acggtcacca ggcagaacac ccggggaccc ctgaggcggc
     2161 aggagacgga gaacaagtac gagaccgacc tgggccgaga ccggaaaggc gatgacaaga
     2221 agaacatgct gatcgacatc atggacacgt cccagcagaa gtcggctggc ctgctgatgg
     2281 tgcacaccgt ggacgccact aagctggaca acgccctgca ggaagaggac gagaaggcag
     2341 aggtggagat gaagccagac agctcgccgt ccgaggtgcc agaaggtgtt tccgaaaccg
     2401 aaggtgcttt acagatctcc gctgcccccg agcccaccac cgtgcccggc agaaccatcg
     2461 tcgcggtggg ctccatggaa gaggcggtga ttttgccatt ccgcatccct cctccccctc
     2521 tggcatccgt ggacttggat gaggatttta tttttacaga gccattgcct cctcccctgg
     2581 aatttgcaaa tagttttgat atccccgatg accgggcagc ttctgtcccg gctctctcag
     2641 acttagtgaa gcagaagaaa agcgacaccc ctcagtcccc ttcgttgaac tccagccaac
     2701 caaccaactc tgcagacagc aagaagccag ccagtctttc aaactgtctg cctgcctcat
     2761 tcctgccacc ccctgaaagc tttgacgccg tcgccgactc tgggatcgag gaggtggaca
     2821 gccggagtag cagcgaccac cacctcgaga cgaccagcac tatctccacc gtgtctagca
     2881 tctccaccct gtcttccgaa ggtggagaga atgtggacac ctgcacagtc tatgcagatg
     2941 ggcaagcatt tatggttgac aaacccccag tacctcctaa gccaaaaatg aagcccatca
     3001 ttcacaaaag caatgcactt tatcaagacg cgctcgtgga agaagatgta gatagctttg
     3061 ttatcccccc gcccgctccc ccgcccccgc cgggcagtgc ccagcctggg atggccaagg
     3121 ttctccagcc aaggacctcc aagttgtggg gcgacgtcac agagatcaaa agcccgattc
     3181 tctcaggccc aaaggcaaac gttattagtg aattgaactc tatcctacag caaatgaacc
     3241 gagagaaatt ggcaaagccg ggggaaggac tggattcacc aatgggagcc aagtccgcca
     3301 gcctcgctcc aagaagcccg gagatcatga gcaccatctc aggtacacgg agcacgacgg
     3361 tcaccttcac tgttcgcccc ggcacctccc agcccatcac cctgcagagc cggccccccg
     3421 actatgaaag caggacctca ggaacaagac gtgccccaag ccctgtggtc tcgccaacag
     3481 agatgaacaa agagaccctg cccgcccccc tgtctgctgc caccgcctct ccttctcccg
     3541 ctctctcaga tgtctttagc cttccaagcc agcccccttc tggggatcta tttggcttga
     3601 acccagcggg acgcagtagg tcgccatccc cctcgatact gcaacagcca atctcaaata
     3661 agccttttac aactaaacct gtccacctgt ggactaaacc agatgtggcc gattggctgg
     3721 aaagtctaaa cttgggtgaa cataaagagg ccttcatgga caatgagatc gatggcagtc
     3781 acttaccaaa cctgcagaag gaggacctca tcgatcttgg ggtaactcga gtcgggcaca
     3841 gaatgaacat agaaagggct ttgaaacagc tgctggacag atgtacagac tacaaagacc
     3901 atgacggtga ttataaagat catgacatcg actacaagga tgacgatgac aagtaactcg
     3961 agataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     4021 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     4081 ccgtatggct ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga
     4141 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     4201 ttccgtggtg tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccatctagct
     4261 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     4321 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     4381 ttttttaaag atctggagac cggtgtggca cgtgcggacc gagcggccgc aggaacccct
     4441 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4501 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4561 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4621 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4681 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4741 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4801 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4861 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4921 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4981 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     5041 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5101 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5161 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5221 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5281 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5341 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5401 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5461 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5521 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5581 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5641 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5701 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5761 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5821 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5881 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5941 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6001 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6061 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6121 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6181 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6241 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6301 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6361 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6421 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6481 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6541 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6601 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6661 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6721 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6781 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6841 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6901 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6961 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7021 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7081 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7141 gccttttgct ggccttttgc tcacatgt
//
