LOCUS       sequence-491437-        7218 bp    DNA     circular SYN 03-NOV-2025
DEFINITION  3' AAVLINK plasmid for SCN8A expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7218)
  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  JOURNAL   Unpublished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 7218)
  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            "Unpublished"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'-SCN8A-FLAG
FEATURES             Location/Qualifiers
     source          1..7218
                     /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             3938..4003
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4068..4088)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4481..4621
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4481..4610
                     /label=AAV2 ITR
     rep_origin      4696..5151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4783..4802)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4993..5014
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5168..5187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5287..5309
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5347..5365)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5433..5537
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5538..5606,5607..6398)
                     /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(5756..5775)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6569..7157
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7058..7077
                     /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 ggttgtgtcc agcggttcaa
     1501 gtgctgccag gtcaacatcg aggaagggct aggcaagtct tggtggatcc tgcggaaaac
     1561 ctgcttcctc atcgtggagc acaactggtt tgagaccttc atcatcttca tgattctgct
     1621 gagcagtggc gccctggcct tcgaggacat ctacattgag cagagaaaga ccatccgcac
     1681 catcctggaa tatgctgaca aagtcttcac ctatatcttc atcctggaga tgttgctcaa
     1741 gtggacagcc tatggcttcg tcaagttctt caccaatgcc tggtgttggc tggacttcct
     1801 cattgtggct gtctctttag tcagccttat agctaatgcc ctgggctact cggaactagg
     1861 tgccataaag tcccttagga ccctaagagc tttgagaccc ttaagagcct tatcacgatt
     1921 tgaagggatg agggtggtgg tgaatgcctt ggtgggcgcc atcccctcca tcatgaatgt
     1981 gctgctggtg tgtctcatct tctggctgat tttcagcatc atgggagtta acttgtttgc
     2041 gggaaagtac cactactgct ttaatgagac ttctgaaatc cgatttgaaa ttgaagatgt
     2101 caacaataaa actgaatgtg aaaagcttat ggaggggaac aatacagaga tcagatggaa
     2161 gaacgtgaag atcaactttg acaatgttgg ggcaggatac ctggcccttc ttcaagtagc
     2221 aaccttcaaa ggctggatgg acatcatgta tgcagctgta gattcccgga agcctgatga
     2281 gcagcctaag tatgaggaca atatctacat gtacatctat tttgtcatct tcatcatctt
     2341 cggctccttc ttcaccctga acctgttcat tggtgtcatc attgataact tcaatcaaca
     2401 aaagaaaaag ttcggaggtc aggacatctt catgaccgaa gaacagaaga agtactacaa
     2461 tgccatgaaa aagctgggct caaagaagcc acagaaacct attccccgcc ccttgaacaa
     2521 aatccaagga atcgtctttg attttgtcac tcagcaagcc tttgacattg ttatcatgat
     2581 gctcatctgc cttaacatgg tgacaatgat ggtggagaca gacactcaaa gcaagcagat
     2641 ggagaacatc ctctactgga ttaacctggt gtttgttatc ttcttcacct gtgagtgtgt
     2701 gctcaaaatg tttgcgttga ggcactacta cttcaccatt ggctggaaca tcttcgactt
     2761 cgtggtagtc atcctctcca ttgtgggaat gttcctggca gatataattg agaaatactt
     2821 tgtttcccca accctattcc gagtcatccg attggcccgt attgggcgca tcttgcgtct
     2881 gatcaaaggc gccaaaggga ttcgtaccct gctctttgcc ttaatgatgt ccttgcctgc
     2941 cctgttcaac atcggccttc tgctcttcct ggtcatgttc atcttctcca tttttgggat
     3001 gtccaatttt gcatatgtga agcacgaggc tggtatcgat gacatgttca actttgagac
     3061 atttggcaac agcatgatct gcctgtttca aatcacaacc tcagctggtt gggatggcct
     3121 gctgctgccc atcctaaacc gcccccctga ctgcagccta gataaggaac acccagggag
     3181 tggctttaag ggagattgtg ggaacccctc agtgggcatc ttcttctttg taagctacat
     3241 catcatctct ttcctaattg tcgtgaacat gtacattgcc atcatcctgg agaacttcag
     3301 tgtagccaca gaggaaagtg cagaccctct gagtgaggat gactttgaga ccttctatga
     3361 gatctgggag aagttcgacc ccgatgccac ccagttcatt gagtactgta agctggcaga
     3421 ctttgcagat gccttggagc atcctctccg agtgcccaag cccaatacca ttgagctcat
     3481 cgctatggat ctgccaatgg tgagcgggga tcgcatccac tgcttggaca tcctttttgc
     3541 cttcaccaag cgggtcctgg gagatagcgg ggagttggac atcctgcggc agcagatgga
     3601 agagcggttc gtggcatcca atccttccaa agtgtcttac gagccaatca caaccacact
     3661 gcgtcgcaag caggaggagg tatctgcagt ggtcctgcag cgtgcctacc ggggacattt
     3721 ggcaaggcgg ggcttcatct gcaaaaagac aacttctaat aagctggaga atggaggcac
     3781 acaccgggag aaaaaagaga gcaccccatc tacagcctcc ctcccgtcct atgacagtgt
     3841 aactaaacct gaaaaggaga aacagcagcg ggcagaggaa ggaagaaggg aaagagccaa
     3901 aagacaaaaa gaggtcagag aatccaagtg ttgtacagac tacaaagacc atgacggtga
     3961 ttataaagat catgacatcg actacaagga tgacgatgac aagtaactcg agataatcaa
     4021 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     4081 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     4141 ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga actcatcgcc
     4201 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     4261 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccatctagct ttatttgtga
     4321 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     4381 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
     4441 atctggagac cggtgtggca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     4501 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     4561 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     4621 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     4681 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4741 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     4801 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4861 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4921 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4981 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     5041 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     5101 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     5161 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     5221 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5281 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     5341 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     5401 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5461 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5521 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5581 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5641 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5701 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5761 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5821 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5881 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5941 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6001 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6061 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6121 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6181 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6241 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6301 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6361 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6421 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6481 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6541 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6601 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6661 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6721 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6781 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6841 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6901 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6961 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7021 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7081 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7141 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7201 ggccttttgc tcacatgt
//
