LOCUS       sequence-481050-        7179 bp    DNA     circular SYN 25-AUG-2025
DEFINITION  3' AAVLINK plasmid for SHANK2 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7179)
  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  JOURNAL   Unpublished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 7179)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 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            "Unpublished"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..7179
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     CDS             288..1319
                     /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             3899..3964
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4029..4049)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4442..4582
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4442..4571
                     /label=AAV2 ITR
     rep_origin      4657..5112
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4744..4763)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4954..4975
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5129..5148)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5248..5270
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5308..5326)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5394..5498
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5499..5567,5568..6359)
                     /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(5717..5736)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6530..7118
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7019..7038
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtcgctc tgcgcgaggc ggctccgccg
      181 cggcggaggg atacggcgca ccatatatat atcgcggggc gcagactcgc gctccggcag
      241 tggtgctggg agtgtcgtgg acgccgtgcc gttactcgta cgccaccatg tccaatttac
      301 tgaccgtaca ccaaaatttg cctgcattac cggtcgatgc aacgagtgat gaggttcgca
      361 agaacctgat ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc
      421 ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc
      481 ccgcagaacc tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag
      541 taaaaactat ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tccgggctgc
      601 cacgaccaag tgacagcaat gctgtttcac tggttatgcg gcggatccga aaagaaaacg
      661 ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg
      721 ttcgttcact catggaaaat agcgatcgct gccaggatat acgtaatctg gcatttctgg
      781 ggattgctta taacaccctg ttacgtatag ccgaaattgc caggatcagg gttaaagata
      841 tctcacgtac tgacggtggg agaatgttaa tccatattgg cagaacgaaa acgctggtta
      901 gcaccgcagg tgtagagaag gcacttagcc tgggggtaac taaactggtc gagcgatgga
      961 tttccgtctc tggtgtagct gatgatccga ataactacct gttttgccgg gtcagaaaaa
     1021 atggtgttgc cgcgccatct gccaccagcc agctatcaac tcgcgccctg gaagggattt
     1081 ttgaagcaac tcatcgattg atttacggcg ctaaggatga ctctggtcag agatacctgg
     1141 cctggtctgg acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt
     1201 caataccgga gatcatgcaa gctggtggct ggaccaatgt aaatattgta atgaactata
     1261 tccgtaacct ggatagtgaa acaggggcaa tggtgcgcct gctggaagat ggcgattagg
     1321 taagtatcaa ggttacaaga caggtttaag gagaccaata actagtataa cttcgtatag
     1381 gatactttat agcaatttat gaaactgggc ttgtcgagac agagaagact cttgcgtttc
     1441 tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac agggcagcag
     1501 catggagatc gacccccagg ccccggagcc accgagccag ctgcggcctg acgaaagcct
     1561 gaccgtcagc agcccctttg ccgccgccat cgccggagcc gtccgcgacc gtgagaagcg
     1621 gctggaagcc aggaggaact ccccggcctt cctctccaca gacctggggg atgaggatgt
     1681 gggcctgggg ccacccgccc ccaggacgcg gccctccatg ttccccgagg agggggattt
     1741 tgctgacgag gacagcgctg agcagctgtc atcccccatg ccgagtgcca cgcccaggga
     1801 gcccgaaaac catttcgtgg gtggcgccga ggccagtgct ccgggtgagg ctgggaggcc
     1861 gctgaattcc acgtccaaag cccaggggcc cgagagcagc ccagcagtgc cctccgcgag
     1921 cagcggcaca gccggccccg ggaattatgt ccacccactc acagggcggc tgcttgatcc
     1981 cagctccccg ctggccctgg cactctccgc aagggaccga gccatgaagg agtctcaaca
     2041 gggacccaaa ggggaggccc ccaaggccga cctcaacaaa cctctttaca ttgataccaa
     2101 aatgcggccc agcctggatg ccggcttccc tacggtcacc aggcagaaca cccggggacc
     2161 cctgaggcgg caggagacgg agaacaagta cgagaccgac ctgggccgag accggaaagg
     2221 cgatgacaag aagaacatgc tgatcgacat catggacacg tcccagcaga agtcggctgg
     2281 cctgctgatg gtgcacaccg tggacgccac taagctggac aacgccctgc aggaagagga
     2341 cgagaaggca gaggtggaga tgaagccaga cagctcgccg tccgaggtgc cagaaggtgt
     2401 ttccgaaacc gaaggtgctt tacagatctc cgctgccccc gagcccacca ccgtgcccgg
     2461 cagaaccatc gtcgcggtgg gctccatgga agaggcggtg attttgccat tccgcatccc
     2521 tcctccccct ctggcatccg tggacttgga tgaggatttt atttttacag agccattgcc
     2581 tcctcccctg gaatttgcaa atagttttga tatccccgat gaccgggcag cttctgtccc
     2641 ggctctctca gacttagtga agcagaagaa aagcgacacc cctcagtccc cttcgttgaa
     2701 ctccagccaa ccaaccaact ctgcagacag caagaagcca gccagtcttt caaactgtct
     2761 gcctgcctca ttcctgccac cccctgaaag ctttgacgcc gtcgccgact ctgggatcga
     2821 ggaggtggac agccggagta gcagcgacca ccacctcgag acgaccagca ctatctccac
     2881 cgtgtctagc atctccaccc tgtcttccga aggtggagag aatgtggaca cctgcacagt
     2941 ctatgcagat gggcaagcat ttatggttga caaaccccca gtacctccta agccaaaaat
     3001 gaagcccatc attcacaaaa gcaatgcact ttatcaagac gcgctcgtgg aagaagatgt
     3061 agatagcttt gttatccccc cgcccgctcc cccgcccccg ccgggcagtg cccagcctgg
     3121 gatggccaag gttctccagc caaggacctc caagttgtgg ggcgacgtca cagagatcaa
     3181 aagcccgatt ctctcaggcc caaaggcaaa cgttattagt gaattgaact ctatcctaca
     3241 gcaaatgaac cgagagaaat tggcaaagcc gggggaagga ctggattcac caatgggagc
     3301 caagtccgcc agcctcgctc caagaagccc ggagatcatg agcaccatct caggtacacg
     3361 gagcacgacg gtcaccttca ctgttcgccc cggcacctcc cagcccatca ccctgcagag
     3421 ccggcccccc gactatgaaa gcaggacctc aggaacaaga cgtgccccaa gccctgtggt
     3481 ctcgccaaca gagatgaaca aagagaccct gcccgccccc ctgtctgctg ccaccgcctc
     3541 tccttctccc gctctctcag atgtctttag ccttccaagc cagccccctt ctggggatct
     3601 atttggcttg aacccagcgg gacgcagtag gtcgccatcc ccctcgatac tgcaacagcc
     3661 aatctcaaat aagcctttta caactaaacc tgtccacctg tggactaaac cagatgtggc
     3721 cgattggctg gaaagtctaa acttgggtga acataaagag gccttcatgg acaatgagat
     3781 cgatggcagt cacttaccaa acctgcagaa ggaggacctc atcgatcttg gggtaactcg
     3841 agtcgggcac agaatgaaca tagaaagggc tttgaaacag ctgctggaca gatgtacaga
     3901 ctacaaagac catgacggtg attataaaga tcatgacatc gactacaagg atgacgatga
     3961 caagtaactc gagataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     4021 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     4081 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttagttctt
     4141 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     4201 ggcactgaca attccgtggt gtttatttgt gaaatttgtg atgctattgc tttatttgta
     4261 accatctagc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     4321 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     4381 gatgtgggag gttttttaaa gatctggaga ccggtgtggc acgtgcggac cgagcggccg
     4441 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     4501 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     4561 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     4621 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     4681 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4741 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4801 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4861 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4921 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4981 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     5041 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5101 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5161 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5221 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5281 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5341 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5401 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5461 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5521 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5581 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5641 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5701 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5761 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5821 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5881 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5941 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6001 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6061 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6121 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6181 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6241 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6301 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6361 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6421 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6481 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6541 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6601 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6661 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6721 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6781 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6841 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6901 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6961 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7021 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7081 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7141 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
