LOCUS       sequence-476863-        6491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for PHRF1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6491)
  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  JOURNAL   Unpublished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 6491)
  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            "Unpublished"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' PHRF1
FEATURES             Location/Qualifiers
     source          1..6491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1976..2041
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2106..2126)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2519..2659
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2519..2648
                     /label=AAV2 ITR
     rep_origin      2734..3189
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2821..2840)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3031..3052
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3206..3225)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3325..3347
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3385..3403)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3471..3575
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3576..3644,3645..4436)
                     /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(3794..3813)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4607..5195
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5096..5115
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5257..5386
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             join(5533..6491,1..73)
                     /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 tgtaatgaac tatatccgta acctggatag tgaaacaggg gcaatggtgc gcctgctgga
       61 agatggcgat taggtaagta tcaaggttac aagacaggtt taaggagacc aataactagt
      121 ataacttcgt ataggatact ttatagcaat ttatgaaact gggcttgtcg agacagagaa
      181 gactcttgcg tttctgatag gcacctattg gtcttactga catccacttt gcctttctct
      241 ccacagggag cacaggcggg gcccctgggg ccacagccgg aggacgtccc ggtcgcggtc
      301 ggggagccct ggcagctctt cctatgagca ctatgagagt aggaagaaga agaaaaggag
      361 atcagcgtcc agacctcggg gaagggagtg ctcccccacc agcagcctgg agaggctctg
      421 caggcacaag catcagcggg aacgcagcca cgagcggcca gacaggaagg agagtgtggc
      481 gtggccccga gaccggagga agcggaggtc ccggtcccca agctcggagc acagggcacg
      541 ggagcacagg cggcctcggt cccgtgagaa gtggccgcag acccggtccc attccccaga
      601 gaggaagggg gctgtgaggg aggcttcccc agcacccctt gcacaggggg agccagggcg
      661 ggaagacctc cccaccaggt tgccagcctt gggggaagca catgtctcgc cggaggtggc
      721 tacggccgac aaggcccccc tgcaggctcc ccctgtcctg gaggtggcag ctgagtgtga
      781 gccggacgac ctggacctgg attatggcga ctccgtggag gccggacacg tctttgatga
      841 tttctcaagc gacgccgttt tcatccagct cgatgacatg agctcgccac cttctcccga
      901 aagcacagac tcttccccag agcgagactt cccactgaag cctgcgttgc ccccagccag
      961 cctggccgtg gccgccatcc agagggaggt gtcattgatg cacgatgaag acccttcgca
     1021 gcccccaccc ctgccagagg gcacccagga gccacatttg ctcaggccgg acgcggctga
     1081 gaaggctgag gcacccagtt ccccggatgt ggcgcctgcg gggaaggaag acagcccctc
     1141 tgcgagtggg agggtacagg aggcagcccg gcctgaggag gtggtttcgc agacccccct
     1201 gctgcggtcc agagccctgg tgaagcgggt cacctggaac ctgcaggagt cggagagcag
     1261 cgcccccgcc gaggacagag ccccccgggc accacttcac aggccacaga agccccgaga
     1321 aggagcctgg gacatggagg atgtggcccc cacaggggtc aggcaggcgt tctccgagct
     1381 gccctttccc agtcacgtgc ttccggaacc cgggttccca gacacagacc cctctcaggt
     1441 ttacagcccc ggcctgccgc ctgccccggc ccagccctca agcatcccac cctgcgcact
     1501 ggtcagccag cccacggtcc agttcatcct tcaggggagc ctgccgctag tgggctgtgg
     1561 ggcagcacag accctggccc cagtgcccgc tgccctgacc ccagcctcag agccagccag
     1621 tcaagccact gcagccagca actcggagga gaagaccccg gcccccaggc tagctgcgga
     1681 gaaaaccaag aaggaggagt acatgaagaa gctgcacatg caggagcgtg ctgtggagga
     1741 ggtgaagctg gccatcaagc ccttctacca gaagagggag gtgaccaagg aggagtacaa
     1801 ggacatcctg cgcaaggccg tgcagaagat ctgccacagc aagagtggag agatcaaccc
     1861 cgtgaaggtg gccaacctgg tgaaggcgta cgtggacaag tacaggcaca tgcgcaggca
     1921 caagaaacca gaggccgggg aggagccgcc cacgcagggg gccgagggct gtacagacta
     1981 caaagaccat gacggtgatt ataaagatca tgacatcgac tacaaggatg acgatgacaa
     2041 gtaactcgag ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     2101 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     2161 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt agttcttgcc
     2221 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     2281 actgacaatt ccgtggtgtt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     2341 atctagcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     2401 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat
     2461 gtgggaggtt ttttaaagat ctggagaccg gtgtggcacg tgcggaccga gcggccgcag
     2521 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     2581 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     2641 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2701 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     2761 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     2821 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2881 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2941 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3001 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3061 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     3121 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     3181 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3241 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3301 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3361 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3421 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3481 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3541 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3601 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3661 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3721 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3781 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3841 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3901 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3961 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4021 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4081 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4141 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4201 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4261 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4321 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4381 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4441 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4501 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4561 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4621 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4681 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4741 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4801 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4861 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4921 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4981 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5041 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5101 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5161 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5221 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
     5281 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     5341 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg ccgcacgcgt
     5401 cgctctgcgc gaggcggctc cgccgcggcg gagggatacg gcgcaccata tatatatcgc
     5461 ggggcgcaga ctcgcgctcc ggcagtggtg ctgggagtgt cgtggacgcc gtgccgttac
     5521 tcgtacgcca ccatgtccaa tttactgacc gtacaccaaa atttgcctgc attaccggtc
     5581 gatgcaacga gtgatgaggt tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg
     5641 ttttctgagc atacctggaa aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc
     5701 aagttgaata accggaaatg gtttcccgca gaacctgaag atgttcgcga ttatcttcta
     5761 tatcttcagg cgcgcggtct ggcagtaaaa accatccagc aacatttggg ccagctaaac
     5821 atgcttcatc gtcggtccgg gctgccacga ccaagtgaca gcaatgctgt ttcactggtt
     5881 atgcggcgga tccgaaaaga aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg
     5941 ttcgaacgca ctgatttcga ccaggttcgt tcactcatgg aaaatagcga tcgctgccag
     6001 gatatacgta atctggcatt tctggggatt gcttataaca ccctgttacg tatagccgaa
     6061 attgccagga tcagggttaa agatatctca cgtactgacg gtgggagaat gttaatccat
     6121 attggcagaa cgaaaacgct ggttagcacc gcaggtgtag agaaggcact tagcctgggg
     6181 gtaactaaac tggtcgagcg atggatttcc gtctctggtg tagctgatga tccgaataac
     6241 tacctgtttt gccgggtcag aaaaaatggt gttgccgcgc catctgccac cagccagcta
     6301 tcaactcgcg ccctggaagg gatttttgaa gcaactcatc gattgattta cggcgctaag
     6361 gatgactctg gtcagagata cctggcctgg tctggacaca gtgcccgtgt cggagccgcg
     6421 cgagatatgg cccgcgctgg agtttcaata ccggagatca tgcaagctgg tggctggacc
     6481 aatgtaaata t
//
