LOCUS       sequence-491433-        6922 bp    DNA     circular SYN 03-NOV-2025
DEFINITION  3' AAVLINK plasmid for RAI1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6922)
  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  JOURNAL   Unpublished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 6922)
  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            "Unpublished"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'-RAI1-FLAG
FEATURES             Location/Qualifiers
     source          1..6922
                     /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             3642..3707
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3772..3792)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4185..4325
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4185..4314
                     /label=AAV2 ITR
     rep_origin      4400..4855
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4487..4506)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4697..4718
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4872..4891)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4991..5013
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5051..5069)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5137..5241
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5242..5310,5311..6102)
                     /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(5460..5479)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6273..6861
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6762..6781
                     /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 ggtctccagc agcccccaga
     1501 aggagggcag ggtgagccag cgggcaaggg tccccaaacc tggtgcaggc agcaagctct
     1561 ctgaccggcc cctccatgcg ctcaaaagga agtcggcctt catggcgccg gtccccacca
     1621 agaagcggaa cctggtcttg cggagccgca gcagcagcag cagcaacgcc agtggcaatg
     1681 ggggagatgg gaaggaggag aggcctgagg gttcccccac cctcttcaag aggatgtctt
     1741 ctcccaagaa agccaagccc accaagggca atggcgagcc tgccacaaag ctcccacccc
     1801 cggagacccc cgatgcctgc ctcaagctcg cctctcgggc agccttccag ggggccatga
     1861 agaccaaggt gctgccaccc cggaagggcc ggggcctgaa gctggaagcc atcgtgcaga
     1921 agatcacctc gcccagcctc aagaagttcg catgtaaagc gccaggggcc tctcctggta
     1981 atcctctgag cccatccctt tccgacaaag accgtgggct caagggtgct gggggcagcc
     2041 cagtgggggt ggaagaaggc ctggtaaatg tgggcaccgg gcagaagctc ccaacttctg
     2101 gggctgatcc gttatgcaga aatccaacca acagatcctt aaaaggcaaa ctcatgaaca
     2161 gtaagaaact gtcttctact gactgtttca aaaccgaggc cttcacatcc ccggaggccc
     2221 tgcagcctgg ggggactgcc ctggcgccta agaagaggag ccggaaaggc cgggcagggg
     2281 cccatggact ctccaaaggc ccgctggaga agcggcccta tcttggcccg gctctgctcc
     2341 tgactccccg agacagggcc agtggcacac aaggggccag tgaggacaac tctggtggag
     2401 gaggcaagaa gccaaagatg gaggagctgg gcctggcctc ccagcccccg gagggcaggc
     2461 cctgccagcc ccagacaagg gcacagaaac agccaggcca caccaactac agcagctatt
     2521 ccaagcggaa gcgcctcact cggggccggg ccaagaacac cacctcttca ccctgtaagg
     2581 ggcgtgccaa gcgacgacga cagcagcagg tgctgcccct ggatcccgca gagcctgaaa
     2641 tccgcctcaa gtacatttcc tcttgcaagc ggctgaggtc agacagccgg acccccgcct
     2701 tctcaccctt cgtgcgggtg gagaagcgag acgcgttcac caccatatgc actgttgtca
     2761 actcccctgg agatgcgccc aagccccaca ggaagccttc ctcctctgcc tcctcttcct
     2821 catcctcgtc ctcgttctcc ttggatgcag ccggggcctc cctggccaca ctccctggag
     2881 gctccatcct gcagccgcgg ccctccttgc ccctctcctc cacgatgcac ttggggcctg
     2941 tggtttccaa ggccctgagt acctcttgcc ttgtttgctg cctctgccaa aacccggcca
     3001 acttcaagga ccttggggac ctctgtgggc cctactaccc tgaacactgc ctccccaaaa
     3061 agaagccaaa actcaaggag aaggtgcggc cagaaggcac ctgtgaggag gcctcgctgc
     3121 cgcttgagag aacactcaaa ggtcccgagt gtgcagctgc cgccactgcc gggaagcccc
     3181 ccaggcctga cggcccagct gacccggcca agcagggccc actgcgcacc agtgcccggg
     3241 gcctgtcccg gaggctgcag agctgctact gctgtgatgg ccgggaggat gggggcgagg
     3301 aggcagcccc agccgacaag ggtcgcaaac atgagtgcag caaggaggct ccggcagagc
     3361 ccggcgggga ggcccaggag cactgggtgc atgaggcctg tgccgtgtgg accggcggcg
     3421 tctacctggt ggccgggaag ctctttgggc tgcaggaggc catgaaggtg gccgtggaca
     3481 tgatgtgttc cagctgccaa gaagccgggg ccaccatcgg gtgctgccac aaaggatgcc
     3541 tccacaccta ccactacccg tgtgccagcg atgcaggttg catattcatc gaagagaact
     3601 tttctttgaa atgtcccaaa cataagaggc tgccgtgtac agactacaaa gaccatgacg
     3661 gtgattataa agatcatgac atcgactaca aggatgacga tgacaagtaa ctcgagataa
     3721 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3781 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3841 ggctttcatt ttctcctcct tgtataaatc ctggttagtt cttgccacgg cggaactcat
     3901 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     3961 ggtgtttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatct agctttattt
     4021 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     4081 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     4141 aaagatctgg agaccggtgt ggcacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     4201 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4261 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     4321 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4381 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4441 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     4501 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4561 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     4621 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4681 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     4741 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     4801 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     4861 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     4921 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4981 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5041 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5101 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5161 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5221 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5281 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5341 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5401 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5461 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5521 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5581 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5641 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5701 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5761 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5821 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5881 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5941 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6001 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6061 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6121 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6181 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6241 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6301 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6361 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6421 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6481 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6541 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6601 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6661 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6721 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6781 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6841 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6901 tgctggcctt ttgctcacat gt
//
