LOCUS       sequence-476871-        7245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for PRR12 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7245)
  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  JOURNAL   Unpublished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 7245)
  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            "Unpublished"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' PRR12
FEATURES             Location/Qualifiers
     source          1..7245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(307..1338)
                     /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   1485..1614
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(1676..2264)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1756..1775)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2435..3226,3227..3295))
                     /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     3058..3077
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3296..3400)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3468..3486
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3524..3546)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3646..3665
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3682..4137)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3819..3840)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4031..4050
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   4212..4352
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4223..4352
                     /label=AAV2 ITR
     primer_bind     4745..4765
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4842..4907)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6804..6815
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             7213..7221
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
ORIGIN
        1 gtcaggctcg agtccaggct gttgtggtca gtgcctgata tggcatcacc cgatgaggtt
       61 cccagaccct cgccagcctt gggcacagac agcttgatct taagtggctt caggggctcc
      121 aaccgggtgc cccctgtgga gagaaaggca aagtggatgt cagtaagacc aataggtgcc
      181 tatcagaaac gcaagagtct tctctgtctc gacaagccca gtttcataaa ttgctataaa
      241 gtatcctata cgaagttata ctagttattg gtctccttaa acctgtcttg taaccttgat
      301 acttacctaa tcgccatctt ccagcaggcg caccattgcc cctgtttcac tatccaggtt
      361 acggatatag ttcattacaa tatttacatt ggtccagcca ccagcttgca tgatctccgg
      421 tattgaaact ccagcgcggg ccatatctcg cgcggctccg acacgggcac tgtgtccaga
      481 ccaggccagg tatctctgac cagagtcatc cttagcgccg taaatcaatc gatgagttgc
      541 ttcaaaaatc ccttccaggg cgcgagttga tagctggctg gtggcagatg gcgcggcaac
      601 accatttttt ctgacccggc aaaacaggta gttattcgga tcatcagcta caccagagac
      661 ggaaatccat cgctcgacca gtttagttac ccccaggcta agtgccttct ctacacctgc
      721 ggtgctaacc agcgttttcg ttctgccaat atggattaac attctcccac cgtcagtacg
      781 tgagatatct ttaaccctga tcctggcaat ttcggctata cgtaacaggg tgttataagc
      841 aatccccaga aatgccagat tacgtatatc ctggcagcga tcgctatttt ccatgagtga
      901 acgaacctgg tcgaaatcag tgcgttcgaa cgctagagcc tgttttgcac gttcaccggc
      961 atcaacgttt tcttttcgga tccgccgcat aaccagtgaa acagcattgc tgtcacttgg
     1021 tcgtggcagc ccggaccgac gatgaagcat gtttagctgg cccaaatgtt gctggatggt
     1081 ttttactgcc agaccgcgcg cctgaagata tagaagataa tcgcgaacat cttcaggttc
     1141 tgcgggaaac catttccggt tattcaactt gcaccatgcc gcccacgacc ggcaaacgga
     1201 cagaagcatt ttccaggtat gctcagaaaa cgcctggcga tccctgaaca tgtccatcag
     1261 gttcttgcga acctcatcac tcgttgcatc gaccggtaat gcaggcaaat tttggtgtac
     1321 ggtcagtaaa ttggacatgg tggcgtacga gtaacggcac ggcgtccacg acactcccag
     1381 caccactgcc ggagcgcgag tctgcgcccc gcgatatata tatggtgcgc cgtatccctc
     1441 cgccgcggcg gagccgcctc gcgcagagcg acgcgtgcgg ccgcaggaac ccctagtgat
     1501 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     1561 cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggacatgt
     1621 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1681 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     1741 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1801 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1861 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1921 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1981 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2041 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2101 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2161 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     2221 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2281 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2341 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2401 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     2461 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     2521 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     2581 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     2641 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     2701 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     2761 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     2821 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     2881 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     2941 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3001 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3061 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     3121 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     3181 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     3241 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     3301 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     3361 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     3421 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     3481 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     3541 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     3601 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     3661 ttgtactgag agtgcaccat aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa
     3721 atttttgtta aatcagctca ttttttaacc aatagaccga aatcggcaaa atcccttata
     3781 aatcaaaaga atagcccgag atagagttga gtgttgttcc agtttggaac aagagtccac
     3841 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     3901 cactacgtga accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     3961 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     4021 cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa ggcgctggca agtgtagcgg
     4081 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtact
     4141 atggttgctt tgacgtatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     4201 catcaggcgc ccctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa
     4261 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
     4321 gggagtggcc aactccatca ctaggggttc ctgcggccgc tcggtccgca cgtgccacac
     4381 cggtctccag atctttaaaa aacctcccac atctccccct gaacctgaaa cataaaatga
     4441 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4501 gcatcacaaa tttcacaaat aaagctagat ggttacaaat aaagcaatag catcacaaat
     4561 ttcacaaata aacaccacgg aattgtcagt gcccaacagc cgagcccctg tccagcagcg
     4621 ggcaaggcag gcggcgatga gttccgccgt ggcaagaact aaccaggatt tatacaagga
     4681 ggagaaaatg aaagccatac gggaagcaat agcatgatac aaaggcatta aagcagcgta
     4741 tccacatagc gtaaaaggag caacatagtt aagaatacca gtcaatcttt cacaaatttt
     4801 gtaatccaga ggttgattat ctcgagtcat cagaattctt acttgtcatc gtcatccttg
     4861 tagtcgatgt catgatcttt ataatcaccg tcatggtctt tgtagtctgt acacccgcag
     4921 cggctgtggg cctgtgcttg ggccagcagg tcactgaagg agtcaaagag ctgttccagc
     4981 cacggctggt tccgcatgca ctgctggacc acctcctcct ggcccaggtc ctcggcgccg
     5041 ccctcgatgg ccagggtctg gtcccggcag cgcaggaagg tgtggtattt atagtggtgg
     5101 agcgaatggt agagtgtata gtatcccgac ttttccagct caaccttgaa ctcctgggct
     5161 ctgaccacgc tcctcttgag cttgctgagc gtcttgcggt tgtagggttc cccagcaggc
     5221 cgcagccgca cagccccctc ttccggagag ccctccccac tctgctccac ttgcagctgt
     5281 gggaacgtgt gcagagcatc ctgcagggct gggcttagcg acagccgctt caggactttc
     5341 ttcttgtgct cattcagcag gccgtctatc ttccgcatgg ggggcaggta cagctcgtca
     5401 tgcgtgtcct ccagggcctg gatcatgtct gggtcaagtg ctgtgctcac caacatctcc
     5461 acgtagctcc ggtacatctc ccgcatcgcc ctggttttga gcagacgccc aggtactgcc
     5521 cgctcgtcct cgctgggggc tccaggggag gactccgagt ccgaggagct gcctggtggg
     5581 cccccgcctg ctgtagcgtt gccgcctgcc tccttcagcc atttcttcct cttcttaggg
     5641 ggcggttcag ccttcacctt ggtaggccgg gctttgggca gccgggatgt ggcaggttgt
     5701 cccgtggcga gactccgctc tggccgtgtc tgccgtccgc tggtggcccg ctcaccccgc
     5761 aatggccgct ctccacgtgt caccttctcc ttctcctttt ccttctcaac aggccgcagg
     5821 agggagggct tctcgggggc agggggctca ggcatggccg tctcaggagt ctgctctggg
     5881 ggcttctcag atgtcgtctt ttcaggggtc tcaggcttgg gaggtggtgc tggggcctta
     5941 gggggaggtg cagagctacc cccagcagat acggggttct tggcctgccc agagcgtctg
     6001 actggcactg ggggccgatg ccagggtttc cgagcacaga ccctcacgta gtccttctta
     6061 ttgacatttt ccaggaactt tacatagagg cgctggtacc gattttttgc atccccaaaa
     6121 taccccaaat aattactggt ggcacaaaac tgcctctggc cgtccttgat ctccggagtg
     6181 aatttctgca gaagggcctt ctggactcgc cagatgggtg ggggttcacg ccccgtctgc
     6241 aacgccagct tgagggaagg gatgtcctca gcacggatga cgaactcgtc ccgttcccgg
     6301 aagatgccct ctccgccact ctcgccggcc tcctcgtccg tctccccaca cactgccgcc
     6361 gtctcctgct ttttggccag atgcaggggc cgggtgtccg gcagctcggg gtcttcggga
     6421 gacggggcgg cgggctcctc aggaggagca gccagtgggg cagcggctgg tgggggtggt
     6481 ggtggaggcg agggcatggc tggtggaggt ggcggcggca gcggcggtgg cggtggtgag
     6541 ctgggcgtgg gggccaccag cgggggtggc gagggcaggg ctggctgtgg cggcggaggg
     6601 ggtggcggag gctgaggctg aggagtcggg gcaggaggtg gagtgggtgg gggtttctcc
     6661 tccagcagcg ggggctcggg agtgccattg ctgttggcac tggggagccc cggaagaggg
     6721 ggtggccctg gaactgcagc cgcgggggcc aagggcggcc catctggggt ggaagtgtct
     6781 tttggcccag caaggggtgg gtcatcgagg gcagagatgg cggaggagat gctctcctgc
     6841 aggtctcggt tcttggcgac agagtcttcc tcatccgagg agaaggaggg cagcgtctcg
     6901 aggttccgct tcagctcctc atcaagccgc ttgcagggtg aagggcagcc aagccccaga
     6961 ccctcactct ctgcagcctc cagggcgccc ccaagcccaa aggctccgga aggtggggga
     7021 tgtggcacag caggggtggc agggggctgg ggctgcaggc ctcgggctgg cacagagggt
     7081 ggcacactct tgggagggct gagcggaggc gtcaggcccg cctggtagag tggtgggtgg
     7141 cgcttgcctg acttgaggaa gtccaagaag gaggccatga aacccgactt catctccggc
     7201 tgcttctcct ccacctcctt aatcttggcc tcgatcttct cccga
//
