LOCUS       sequence-476872-        6324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for NINL expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6324)
  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  JOURNAL   Unpublished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 6324)
  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            "Unpublished"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' NINL
FEATURES             Location/Qualifiers
     source          1..6324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(70..135)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1696..1715)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             complement(1871..2902)
                     /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"
     repeat_region   3049..3178
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(3240..3828)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3320..3339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3999..4790,4791..4859))
                     /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     4622..4641
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4860..4964)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5032..5050
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5088..5110)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5210..5229
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5246..5701)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5383..5404)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5595..5614
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5776..5916
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5787..5916
                     /label=AAV2 ITR
     primer_bind     join(6309..6324,1..5)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 caacatagtt aagaatacca gtcaatcttt cacaaatttt gtaatccaga ggttgattat
       61 ctcgagttac ttgtcatcgt catccttgta gtcgatgtca tgatctttat aatcaccgtc
      121 atggtctttg tagtctgtac acacagagag ggctgcgggg gcaatcctac tgacgagttt
      181 gttgagagcg cgaacttttt cttccaagag gcggctttgt ttctcggcgc ctcgctgctt
      241 ctcctcggtg gcctgaagtg ctctcaccag gtgggcgttc tccacgtaca gctccttcag
      301 cagcaggtcg gacttcgtgt tcttttcaaa ctgctccttc agcttatcca ctttctcttg
      361 gaggagcatt tccatattct tcagaatcat ctccgcctct tccacccgct cttctgtggc
      421 tttcagctgt ttctcatgtt cttcccgctg tgcatccagg cgcctcctgg cctgctctcc
      481 ctgaaggctg agcaggcgat gcagctcggc cacacggtcc tggggcacca gccggacctc
      541 ctgcaagtgc tgggcctggg cctgcctcag cctcaggtga gctccctgca cctggtcttg
      601 actttcctca agggtctggg tcagctctga ccatggcagc tgcaggctct gatgttcctg
      661 attcaggcat tcaagttcaa ctctaagttt ttggatttgg tcactctgct gctgcccact
      721 gcgaaccacc tcctccaggc tctgtgttaa catctgaatg gtcacacgat gctcctcgtt
      781 ttgggcctgg tgggtagaag cctcctgtcc cagttgaaga accctgacat tgagctggga
      841 taattgatcc ttgtagttct gattctgtct gttgagcacc tccatctcag agcaggcctt
      901 ttccttgtct ttctttaaaa cctcaatttc cttcctctgt gcatcgtgag tactttctgc
      961 agcttcaagc tcttgccgaa cccttcccag atcgtttttc aacaaagtgt tttcttcaga
     1021 aagtctatgg aactccagtc tgtcgttctc tctcaaatct acatgtttct ccagagcccg
     1081 gacgacgtct tccagatgta gaagtttggt ttccatgtca tccttctctc tctccagcgc
     1141 tattttggtc tccccctctt ctggggcagc aagctgctcc tgccaggaac cctgcgggtc
     1201 tgcgagctgg aggtgggatg gcaaggaccc tctcctggca acctccacac tgtgcttgtg
     1261 acacccaggc tccagggcgc cctcggcccg ggcctgctgc tccgaggcct gctcctgggt
     1321 gcccctgctc cagcttcgtg ctcgctcttc ctgaatggcc tgtagcctct cagcctgtcc
     1381 cctgcacgaa gcggccggcc tcaggggtgg ctcccacatc cgtggctggg tttgcgaggc
     1441 gtctctctct gttcccagca gaggcagctc ccgggctcca ggctgctcca gccccgctgc
     1501 gctcgcgccg aaaggctctg gctcctttgg gacaatatcc ccatccactc cacagggctg
     1561 catgcgtgac cacctctctg aggggccgtg ggatgccggg gcaggggcgg gggccgggct
     1621 ctgcgtagct tctgtgtcct gggcttgcct gcggcgaggc ccggctcctg ccgcctcctc
     1681 agactctctg ccatcacctg tggagagaaa ggcaaagtgg atgtcagtaa gaccaatagg
     1741 tgcctatcag aaacgcaaga gtcttctctg tctcgacaag cccagtttca taaattgcta
     1801 taaagtatcc tatacgaagt tatactagtt attggtctcc ttaaacctgt cttgtaacct
     1861 tgatacttac ctaatcgcca tcttccagca ggcgcaccat tgcccctgtt tcactatcca
     1921 ggttacggat atagttcatt acaatattta cattggtcca gccaccagct tgcatgatct
     1981 ccggtattga aactccagcg cgggccatat ctcgcgcggc tccgacacgg gcactgtgtc
     2041 cagaccaggc caggtatctc tgaccagagt catccttagc gccgtaaatc aatcgatgag
     2101 ttgcttcaaa aatcccttcc agggcgcgag ttgatagctg gctggtggca gatggcgcgg
     2161 caacaccatt ttttctgacc cggcaaaaca ggtagttatt cggatcatca gctacaccag
     2221 agacggaaat ccatcgctcg accagtttag ttacccccag gctaagtgcc ttctctacac
     2281 ctgcggtgct aaccagcgtt ttcgttctgc caatatggat taacattctc ccaccgtcag
     2341 tacgtgagat atctttaacc ctgatcctgg caatttcggc tatacgtaac agggtgttat
     2401 aagcaatccc cagaaatgcc agattacgta tatcctggca gcgatcgcta ttttccatga
     2461 gtgaacgaac ctggtcgaaa tcagtgcgtt cgaacgctag agcctgtttt gcacgttcac
     2521 cggcatcaac gttttctttt cggatccgcc gcataaccag tgaaacagca ttgctgtcac
     2581 ttggtcgtgg cagcccggac cgacgatgaa gcatgtttag ctggcccaaa tgttgctgga
     2641 tggtttttac tgccagaccg cgcgcctgaa gatatagaag ataatcgcga acatcttcag
     2701 gttctgcggg aaaccatttc cggttattca acttgcacca tgccgcccac gaccggcaaa
     2761 cggacagaag cattttccag gtatgctcag aaaacgcctg gcgatccctg aacatgtcca
     2821 tcaggttctt gcgaacctca tcactcgttg catcgaccgg taatgcaggc aaattttggt
     2881 gtacggtcag taaattggac atggtggcgt acgagtaacg gcacggcgtc cacgacactc
     2941 ccagcaccac tgccggagcg cgagtctgcg ccccgcgata tatatatggt gcgccgtatc
     3001 cctccgccgc ggcggagccg cctcgcgcag agcgacgcgt gcggccgcag gaacccctag
     3061 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     3121 aggtcgcccg acgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcaggac
     3181 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3241 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3301 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3361 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3421 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3481 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3541 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3601 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3661 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3721 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3781 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3841 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3901 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3961 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4021 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4081 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4141 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4201 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4261 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4321 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4381 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4441 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4501 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4561 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4621 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4681 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4741 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4801 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4861 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4921 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4981 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     5041 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     5101 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     5161 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     5221 cagattgtac tgagagtgca ccataaaatt gtaaacgtta atattttgtt aaaattcgcg
     5281 ttaaattttt gttaaatcag ctcatttttt aaccaataga ccgaaatcgg caaaatccct
     5341 tataaatcaa aagaatagcc cgagatagag ttgagtgttg ttccagtttg gaacaagagt
     5401 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     5461 ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca
     5521 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     5581 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct ggcaagtgta
     5641 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     5701 tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     5761 accgcatcag gcgcccctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg
     5821 caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca
     5881 gagagggagt ggccaactcc atcactaggg gttcctgcgg ccgctcggtc cgcacgtgcc
     5941 acaccggtct ccagatcttt aaaaaacctc ccacatctcc ccctgaacct gaaacataaa
     6001 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     6061 aatagcatca caaatttcac aaataaagct agatggttac aaataaagca atagcatcac
     6121 aaatttcaca aataaacacc acggaattgt cagtgcccaa cagccgagcc cctgtccagc
     6181 agcgggcaag gcaggcggcg atgagttccg ccgtggcaag aactaaccag gatttataca
     6241 aggaggagaa aatgaaagcc atacgggaag caatagcatg atacaaaggc attaaagcag
     6301 cgtatccaca tagcgtaaaa ggag
//
