LOCUS       sequence-476865-        6799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for KANK1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6799)
  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  JOURNAL   Unpublished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 6799)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 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            "Unpublished"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' KANK1
FEATURES             Location/Qualifiers
     source          1..6799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     75..94
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             2118..2183
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2260..2280)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2673..2813
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2673..2802
                     /label=AAV2 ITR
     rep_origin      2888..3343
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2975..2994)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3185..3206
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3360..3379)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3479..3501
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3539..3557)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3625..3729
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3730..3798,3799..4590)
                     /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(3948..3967)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4761..5349
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5250..5269
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5411..5540
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             5687..6718
                     /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"
ORIGIN
        1 gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac ctattggtct
       61 tactgacatc cactttgcct ttctctccac aggtgcacca gttcaccaac accgagacgg
      121 ccaccctcat agagtcctgc accaacactt gtctaagcac tttggacaag cagaccagca
      181 cccagactgt ggagacgcgg acagtagctg taggagaagg ccgtgtcaag gacatcaact
      241 cctccaccaa gacgcggtcc attggtgttg gaacgttgct ttctggccat tctgggtttg
      301 acaggccatc agctgtgaag accaaagagt caggtgtggg gcagataaat attaacgaca
      361 actatctggt tggtctcaaa atgaggacta tagcttgtgg gccaccacag ttgactgtgg
      421 ggctgacagc cagcagaagg agcgtggggg ttggggatga ccctgtaggg gaatctctgg
      481 agaaccccca gcctcaagct ccacttggaa tgatgactgg cctggatcac tacattgagc
      541 gtatccagaa gctgctggca gaacagcaga cactgctggc tgagaactac agtgaactgg
      601 cagaagcttt cggggaacct cactcacaga tgggctccct caactctcag ctcatcagca
      661 ccctgtcgtc tatcaactct gtcatgaaat ctgcaagcac tgaagagctg aggaaccctg
      721 acttccagaa aaccagtctg ggtaaaatca caggcaatta tttgggatat acctgtaagt
      781 gtgggggcct tcagtcagga agtcccttaa gctcccagac atcccagcct gagcaagaag
      841 tggggacctc agaaggaaag ccaatcagca gcctggatgc cttccccact caggaaggta
      901 cgctgtctcc agtgaacctg acagacgacc agatcgccgc tggcctctat gcatgtacaa
      961 acaatgaaag tacactgaag tccatcatga agaagaaaga tggtaacaaa gattcaaatg
     1021 gcgcaaaaaa gaatcttcag tttgttggca ttaatggagg gtatgaaaca acttcaagtg
     1081 atgattccag ctcagatgaa agctcttctt ccgagtcaga tgacgagtgt gatgtcattg
     1141 agtatcctct tgaagaagag gaggaggagg aggatgaaga cactcgggga atggcagaag
     1201 ggcaccatgc agttaatatt gaaggtttga agtctgccag ggtggaagat gaaatgcagg
     1261 ttcaagaatg tgaacctgag aaggtggaaa tcagagagag gtatgaatta agtgaaaaga
     1321 tgttgtctgc atgcaactta ctgaaaaata ctataaatga ccccaaagct ttgaccagca
     1381 aagatatgag gttctgtctg aacaccctcc agcacgagtg gttccgcgtg tccagtcaga
     1441 agtcagccat tccagccatg gtgggggact acatagctgc ttttgaggcc atttccccag
     1501 atgtcctccg ctatgtcatc aacttggcag acggcaacgg caacacagcc ctccattaca
     1561 gcgtgtccca ctccaacttc gagattgtga agctgctgtt agatgccgat gtgtgtaatg
     1621 tggatcacca gaacaaggca ggctacaccc ccatcatgtt ggcggccctc gccgctgtgg
     1681 aagcagagaa ggacatgcgg attgtggaag aactcttcgg ctgtggggat gtgaatgcca
     1741 aagctagtca ggcgggacag acggccctca tgctggcggt cagtcacgga cggatagaca
     1801 tggtgaaggg ccttctggcc tgtggggctg atgtcaacat ccaggatgac gagggctcca
     1861 cggccctcat gtgtgccagc gagcacgggc acgtggagat tgtcaagctg ctgctggccc
     1921 agcccggctg caacggtcac ctagaggaca acgatggcag cactgcgctc tcaatcgccc
     1981 tggaagcagg acacaaggac atcgctgttc ttctgtatgc ccatgtcaac tttgcaaaag
     2041 cccagtctcc gggcacccct aggcttggaa ggaagacgtc tcctggcccc acccaccgag
     2101 gttcatttga ttgtacagac tacaaagacc atgacggtga ttataaagat catgacatcg
     2161 actacaagga tgacgatgac aagtaagaat tctgatgact caagataatc aacctctgga
     2221 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     2281 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     2341 ctcctccttg tataaatcct ggttagttct tgccacggcg gaactcatcg ccgcctgcct
     2401 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgtttatttg
     2461 tgaaatttgt gatgctattg ctttatttgt aaccatctag ctttatttgt gaaatttgtg
     2521 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     2581 gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa agatctggag
     2641 accggtgtgg cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac
     2701 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     2761 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     2821 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     2881 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2941 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     3001 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3061 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     3121 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3181 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     3241 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     3301 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     3361 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     3421 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3481 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     3541 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     3601 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3661 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3721 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3781 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3841 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3901 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3961 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     4021 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     4081 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     4141 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     4201 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     4261 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     4321 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     4381 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     4441 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     4501 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     4561 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     4621 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4681 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4741 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4801 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4861 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     4921 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4981 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5041 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5101 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5161 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5221 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5281 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5341 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5401 gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg
     5461 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
     5521 ctccatcact aggggttcct gcggccgcac gcgtcgctct gcgcgaggcg gctccgccgc
     5581 ggcggaggga tacggcgcac catatatata tcgcggggcg cagactcgcg ctccggcagt
     5641 ggtgctggga gtgtcgtgga cgccgtgccg ttactcgtac gccaccatgt ccaatttact
     5701 gaccgtacac caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa
     5761 gaacctgatg gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct
     5821 tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc
     5881 cgcagaacct gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt
     5941 aaaaaccatc cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc
     6001 acgaccaagt gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt
     6061 tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt
     6121 tcgttcactc atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg
     6181 gattgcttat aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat
     6241 ctcacgtact gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag
     6301 caccgcaggt gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat
     6361 ttccgtctct ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa
     6421 tggtgttgcc gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt
     6481 tgaagcaact catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc
     6541 ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc
     6601 aataccggag atcatgcaag ctggtggctg gaccaatgta aatattgtaa tgaactatat
     6661 ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattaggt
     6721 aagtatcaag gttacaagac aggtttaagg agaccaataa ctagtataac ttcgtatagg
     6781 atactttata gcaatttat
//
