LOCUS       sequence-476880-        6599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for NEXMIF expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6599)
  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  JOURNAL   Unpublished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 6599)
  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            "Unpublished"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' NEXMIF
FEATURES             Location/Qualifiers
     source          1..6599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2030..2095
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2160..2180)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2573..2713
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2573..2702
                     /label=AAV2 ITR
     rep_origin      2788..3243
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2875..2894)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3085..3106
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3260..3279)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3379..3401
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3439..3457)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3525..3629
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3630..3698,3699..4490)
                     /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(3848..3867)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4661..5249
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5150..5169
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5311..5440
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             join(5587..6599,1..19)
                     /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 gctggaagat ggcgattagg taagtatcaa ggttacaaga caggtttaag gagaccaata
       61 actagtataa cttcgtatag gatactttat agcaatttat gaaactgggc ttgtcgagac
      121 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
      181 ttctctccac agggagatag ccccaaccca atccagtgaa tttggagcct cccaagtagt
      241 ctccatggaa aataacctca cacctacaac atacaatcca atctgtctca atagtggtgg
      301 cagtaattgc aacaaggtcc tgtatgactc catgcaagat acccaactcc catctgatga
      361 ctcttaccaa ttatgtcact ttaataatgg agagatctgc tttcctttcc agcaggggcc
      421 agtcaatatg gatgatggtc ggctctttag ctttgattca atggccccac tctctgtcag
      481 ctcaagtaat tattgctcct taagcttgaa gtcctgtgaa aaggatggcg atgatgatat
      541 cactgatgac ttcctggccc attgcagccc taagctggtg atccagcaaa gcattgatga
      601 gatagcacca ctaaaggagt ccactgacct cctggatata tccaacttca cccctgacaa
      661 attccgccac tcttcccttt cagagatgtc cccaccggac acccctagtc tttcccctca
      721 aattaccaga tgtgagagta tgaagacact aggaacacta aaggggttcc aagagggtgt
      781 cccaggacca ttggacagtg tggaaaaaat caagtgggac tgcagtaccc tttcacggca
      841 ggtccaaatg gaggatggat ttactttaaa taatcaccag tttcagttcc atatgttcaa
      901 tgatgaggat tctgtcagcc tgctccaaaa aaacccttgc ctgtcaacat ttaatgatcc
      961 atctggtcaa attagtacca acaacaaagt gtcaaaatca agaaagaaaa gttcacccag
     1021 caagagtggg gctatgaacc aaagctcttc tcagaaaaac accaggaaaa aatccctcaa
     1081 aggtaacaac aaggggattg aaaaaccacc tggcaaaaac tcccgccagg tccctaagtc
     1141 cacaaagaaa gggaaataca tggctgccat caatggagag aaaatgcaaa ttggcattgg
     1201 ccgtggggga agccaaacca acaccatatc ctccactgga aagacattgg ctgaatgtat
     1261 ccaacatggt ggccctatgg cctctatgaa gatgccaagt caaaagggac tttctggaga
     1321 ttgggccttg gggaaggaga gcagcccagg ctggagtgat atgagcatgg gcaccaacac
     1381 caacagcctt ctggatgatg accaacggga atttcaggag ccttcctata tcttgtccaa
     1441 cattgcctct ggtatggcag atgtgcagag attcatgatg gcctccatag agcccctttg
     1501 ggaacccatg gagcaccatg gggatcccaa catattctac tcccctgagt ccaatagtct
     1561 aaaattaaaa accctcaaaa tattggctgg gacaccacag gagtctaaga aaaagatcaa
     1621 cagtgggtcc caaggagcca ctaagaatca caggtcaatc aagggtgtga gcaaaagcaa
     1681 tgggaaaaca gcaataggtg atcctggtcg tgcaaacatg cctggttata acgaggactc
     1741 tcgctctacc ttctttgata aaaagtatag taacatgagc actttaggca ataacggacc
     1801 gacacacaaa aagttgtatc gtcacaaatc cagctccaag gccctgagag acgagaaatg
     1861 taagggaaag cacatggagc gagaacaggt ccacaaggat gagtctggga cagcttcttt
     1921 tgaaaaactg agggattccg actacaatct cctaaaagca gaaacaacct tttgggtttt
     1981 acctgtgttt gaagaagaga ctcgcatttt ccagaaagac atttgtacag actacaaaga
     2041 ccatgacggt gattataaag atcatgacat cgactacaag gatgacgatg acaagtaact
     2101 cgagataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     2161 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     2221 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttagttct tgccacggcg
     2281 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     2341 aattccgtgg tgtttatttg tgaaatttgt gatgctattg ctttatttgt aaccatctag
     2401 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     2461 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga
     2521 ggttttttaa agatctggag accggtgtgg cacgtgcgga ccgagcggcc gcaggaaccc
     2581 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     2641 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     2701 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     2761 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     2821 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     2881 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2941 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3001 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3061 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     3121 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     3181 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3241 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     3301 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3361 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3421 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     3481 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3541 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3601 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3661 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3721 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3781 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3841 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3901 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3961 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4021 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4081 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     4141 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     4201 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     4261 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     4321 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     4381 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4441 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4501 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4561 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4621 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4681 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4741 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4801 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4861 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4921 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4981 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5041 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5101 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5161 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5221 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5281 tggccttttg ctggcctttt gctcacatgt cctgcaggca gctgcgcgct cgctcgctca
     5341 ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg
     5401 cgcagagagg gagtggccaa ctccatcact aggggttcct gcggccgcac gcgtcgctct
     5461 gcgcgaggcg gctccgccgc ggcggaggga tacggcgcac catatatata tcgcggggcg
     5521 cagactcgcg ctccggcagt ggtgctggga gtgtcgtgga cgccgtgccg ttactcgtac
     5581 gccaccatgt ccaatttact gaccgtacac caaaatttgc ctgcattacc ggtcgatgca
     5641 acgagtgatg aggttcgcaa gaacctgatg gacatgttca gggatcgcca ggcgttttct
     5701 gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg
     5761 aataaccgga aatggtttcc cgcagaacct gaagatgttc gcgattatct tctatatctt
     5821 caggcgcgcg gtctggcagt aaaaaccatc cagcaacatt tgggccagct aaacatgctt
     5881 catcgtcggt ccgggctgcc acgaccaagt gacagcaatg ctgtttcact ggttatgcgg
     5941 cggatccgaa aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa
     6001 cgcactgatt tcgaccaggt tcgttcactc atggaaaata gcgatcgctg ccaggatata
     6061 cgtaatctgg catttctggg gattgcttat aacaccctgt tacgtatagc cgaaattgcc
     6121 aggatcaggg ttaaagatat ctcacgtact gacggtggga gaatgttaat ccatattggc
     6181 agaacgaaaa cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact
     6241 aaactggtcg agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg
     6301 ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact
     6361 cgcgccctgg aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac
     6421 tctggtcaga gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat
     6481 atggcccgcg ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta
     6541 aatattgtaa tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcct
//
