LOCUS       sequence-491762-        7042 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  3' AAVLINK plasmid for CACNA1C expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7042)
  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  JOURNAL   Unpublished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 7042)
  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            "Unpublished"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'-CACNA1C-FLAG
FEATURES             Location/Qualifiers
     source          1..7042
                     /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             3762..3827
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3892..3912)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4305..4445
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4305..4434
                     /label=AAV2 ITR
     rep_origin      4520..4975
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4607..4626)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4817..4838
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4992..5011)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5111..5133
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5171..5189)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5257..5361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5362..5430,5431..6222)
                     /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(5580..5599)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6393..6981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6882..6901
                     /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 ggtgtttggg aaaattgccc
     1501 tgaatgatac cacagagatc aaccggaaca acaactttca gaccttcccc caggccgtgc
     1561 tgctcctctt caggtgtgcc accggggagg cctggcagga catcatgctg gcctgcatgc
     1621 caggcaagaa gtgtgcccca gagtccgagc ccagcaacag cacggagggt gaaacaccct
     1681 gtggtagcag ctttgctgtc ttctacttca tcagcttcta catgctctgt gccttcctga
     1741 tcatcaacct ctttgtagct gtcatcatgg acaactttga ctacctgaca agggactggt
     1801 ccatccttgg tccccaccac ctggatgagt ttaaaagaat ctgggcagag tatgaccctg
     1861 aagccaaggg tcgtatcaaa cacctggatg tggtgaccct cctccggcgg attcagccgc
     1921 cactaggttt tgggaagctg tgccctcacc gcgtggcttg caaacgcctg gtctccatga
     1981 acatgcctct gaacagcgac gggacagtca tgttcaatgc caccctgttt gccctggtca
     2041 ggacggccct gaggatcaaa acagaaggga acctagaaca agccaatgag gagctgcggg
     2101 cgatcatcaa gaagatctgg aagcggacca gcatgaagct gctggaccag gtggtgcccc
     2161 ctgcaggtga tgatgaggtc accgttggca agttctacgc cacgttcctg atccaggagt
     2221 acttccggaa gttcaagaag cgcaaagagc agggccttgt gggcaagccc tcccagagga
     2281 acgcgctgtc tctgcaggct ggcttgcgca cactgcatga catcgggcct gagatccgac
     2341 gggccatctc tggagatctc accgctgagg aggagctgga caaggccatg aaggaggctg
     2401 tgtccgctgc ttctgaagat gacatcttca ggagggccgg tggcctgttc ggcaaccacg
     2461 tcagctacta ccaaagcgac ggccggagcg ccttccccca gaccttcacc actcagcgcc
     2521 cgctgcacat caacaaggcg ggcagcagcc agggcgacac tgagtcgcca tcccacgaga
     2581 agctggtgga ctccaccttc accccgagca gctactcgtc caccggctcc aacgccaaca
     2641 tcaacaacgc caacaacacc gccctgggtc gcctccctcg ccccgccggc taccccagca
     2701 cagtcagcac tgtggagggc cacgggcccc ccttgtcccc tgccatccgg gtgcaggagg
     2761 tggcgtggaa gctcagctcc aacaggtgcc actcccggga gagccaggca gccatggcgg
     2821 gtcaggagga gacgtctcag gatgagacct atgaagtgaa gatgaaccat gacacggagg
     2881 cctgcagtga gcccagcctg ctctccacag agatgctctc ctaccaggat gacgaaaatc
     2941 ggcaactgac gctcccagag gaggacaaga gggacatccg gcaatctccg aagaggggtt
     3001 tcctccgctc tgcctcacta ggtcgaaggg cctccttcca cctggaatgt ctgaagcgac
     3061 agaaggaccg agggggagac atctctcaga agacagtcct gcccttgcat ctggttcatc
     3121 atcaggcatt ggcagtggca ggcctgagcc ccctcctcca gagaagccat tcccctgcct
     3181 cattccctag gccttttgcc accccaccag ccacacctgg cagccgaggc tggcccccac
     3241 agcccgtccc caccctgcgg cttgaggggg tcgagtccag tgagaaactc aacagcagct
     3301 tcccatccat ccactgcggc tcctgggctg agaccacccc cggtggcggg ggcagcagcg
     3361 ccgcccggag agtccggccc gtctccctca tggtgcccag ccaggctggg gccccaggga
     3421 ggcagttcca cggcagtgcc agcagcctgg tggaagcggt cttgatttca gaaggactgg
     3481 ggcagtttgc tcaagatccc aagttcatcg aggtcaccac ccaggagctg gccgacgcct
     3541 gcgacatgac catagaggag atggagagcg cggccgacaa catcctcagc gggggcgccc
     3601 cacagagccc caatggcgcc ctcttaccct ttgtgaactg cagggacgcg gggcaggacc
     3661 gagccggggg cgaagaggac gcgggctgtg tgcgcgcgcg gggtcgaccg agtgaggagg
     3721 agctccagga cagcagggtc tacgtcagca gcctgtgtac agactacaaa gaccatgacg
     3781 gtgattataa agatcatgac atcgactaca aggatgacga tgacaagtaa ctcgagataa
     3841 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3901 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3961 ggctttcatt ttctcctcct tgtataaatc ctggttagtt cttgccacgg cggaactcat
     4021 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     4081 ggtgtttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatct agctttattt
     4141 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     4201 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     4261 aaagatctgg agaccggtgt ggcacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     4321 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4381 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     4441 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4501 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4561 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     4621 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4681 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     4741 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4801 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     4861 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     4921 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     4981 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     5041 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5101 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5161 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5221 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5281 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5341 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5401 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5461 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5521 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5581 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5641 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5701 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5761 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5821 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5881 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5941 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6001 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6061 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6121 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6181 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6241 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6301 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6361 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6421 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6481 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6541 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6601 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6661 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6721 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6781 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6841 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6901 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6961 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7021 tgctggcctt ttgctcacat gt
//
