LOCUS       sequence-476862-        6498 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for KDM2B expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6498)
  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  JOURNAL   Unpublished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 6498)
  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            "Unpublished"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' KDM2B
FEATURES             Location/Qualifiers
     source          1..6498
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1111..1240
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(1302..1890)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1382..1401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2061..2852,2853..2921))
                     /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     2684..2703
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2922..3026)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3094..3112
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3150..3172)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3272..3291
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3308..3763)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3445..3466)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3657..3676
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3838..3978
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3849..3978
                     /label=AAV2 ITR
     primer_bind     4371..4391
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4456..4521)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(join(6431..6498,1..964))
                     /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 ttacaatatt tacattggtc cagccaccag cttgcatgat ctccggtatt gaaactccag
       61 cgcgggccat atctcgcgcg gctccgacac gggcactgtg tccagaccag gccaggtatc
      121 tctgaccaga gtcatcctta gcgccgtaaa tcaatcgatg agttgcttca aaaatccctt
      181 ccagggcgcg agttgatagc tggctggtgg cagatggcgc ggcaacacca ttttttctga
      241 cccggcaaaa caggtagtta ttcggatcat cagctacacc agagacggaa atccatcgct
      301 cgaccagttt agttaccccc aggctaagtg ccttctctac acctgcggtg ctaaccagcg
      361 ttttcgttct gccaatatgg attaacattc tcccaccgtc agtacgtgag atatctttaa
      421 ccctgatcct ggcaatttcg gctatacgta acagggtgtt ataagcaatc cccagaaatg
      481 ccagattacg tatatcctgg cagcgatcgc tattttccat gagtgaacga acctggtcga
      541 aatcagtgcg ttcgaacgct agagcctgtt ttgcacgttc accggcatca acgttttctt
      601 ttcggatccg ccgcataacc agtgaaacag cattgctgtc acttggtcgt ggcagcccgg
      661 accgacgatg aagcatgttt agctggccca aatgttgctg gatggttttt actgccagac
      721 cgcgcgcctg aagatataga agataatcgc gaacatcttc aggttctgcg ggaaaccatt
      781 tccggttatt caacttgcac catgccgccc acgaccggca aacggacaga agcattttcc
      841 aggtatgctc agaaaacgcc tggcgatccc tgaacatgtc catcaggttc ttgcgaacct
      901 catcactcgt tgcatcgacc ggtaatgcag gcaaattttg gtgtacggtc agtaaattgg
      961 acatggtggc gtacgagtaa cggcacggcg tccacgacac tcccagcacc actgccggag
     1021 cgcgagtctg cgccccgcga tatatatatg gtgcgccgta tccctccgcc gcggcggagc
     1081 cgcctcgcgc agagcgacgc gtgcggccgc aggaacccct agtgatggag ttggccactc
     1141 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     1201 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg acatgtgagc aaaaggccag
     1261 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1321 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1381 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1441 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1501 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1561 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1621 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1681 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1741 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1801 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1861 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1921 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1981 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2041 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     2101 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2161 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     2221 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     2281 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     2341 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     2401 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2461 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     2521 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     2581 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     2641 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     2701 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     2761 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     2821 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     2881 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     2941 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3001 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     3061 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     3121 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     3181 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     3241 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     3301 caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     3361 agctcatttt ttaaccaata gaccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     3421 cccgagatag agttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     3481 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     3541 tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     3601 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     3661 aagaaagcga aaggagcggg cgctaaggcg ctggcaagtg tagcggtcac gctgcgcgta
     3721 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg ttgctttgac
     3781 gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgcccct
     3841 gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg
     3901 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
     3961 ccatcactag gggttcctgc ggccgctcgg tccgcacgtg ccacaccggt ctccagatct
     4021 ttaaaaaacc tcccacatct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     4081 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4141 acaaataaag ctagatggtt acaaataaag caatagcatc acaaatttca caaataaaca
     4201 ccacggaatt gtcagtgccc aacagccgag cccctgtcca gcagcgggca aggcaggcgg
     4261 cgatgagttc cgccgtggca agaactaacc aggatttata caaggaggag aaaatgaaag
     4321 ccatacggga agcaatagca tgatacaaag gcattaaagc agcgtatcca catagcgtaa
     4381 aaggagcaac atagttaaga ataccagtca atctttcaca aattttgtaa tccagaggtt
     4441 gattatctcg agttacttgt catcgtcatc cttgtagtcg atgtcatgat ctttataatc
     4501 accgtcatgg tctttgtagt ctgtacaact cagtttttgc aggagttttt cttctacttg
     4561 cccaaactgg acactcacag acatctcggc tatgaactgc tcacagcctt ccttggtgac
     4621 ttgcttgcag tacctcaggt caatatgaca gatgtttcca cagcgtttga agaaggacag
     4681 gcactgatca gtgaccttat tgcagtcaga caggttgatc tcggttaagg agtctcgggt
     4741 ggtggtgcca acagcagtga gcaggttgat agactggtcg gtgacgtggt tacagtaact
     4801 gaggtggagc ttggagagca ggggcatgtg gcggatgatg agccgcaggg aggcatctgt
     4861 gatgtccagg cctgccaggc gcagctccac gatgttccgg agcttgctcc gattgtccat
     4921 ctgacctggc ctgttgtctg tgggcgggga caggagatcc cgcatctggg catcctttag
     4981 tccctccacc cactggacat ccagggtccg gagcagcgga caactggagc tgcaaagggc
     5041 cgagaccgcg atccatgagc agcctgacag caccaagtcc cggagcccag gcagccggtt
     5101 gatgagccag ctcagctgct tcttggagat attggtccag ctgaggtcga gggagacggg
     5161 ctgtcgccgg atgatgccac tcagcatcag gggtgtgata gacttgcagt ggttcaggtc
     5221 aatgcgggtc cacaaccgct tatcgcagca ccagcggttc caggtcctgc agacccgcat
     5281 gcacacacac aggtcttggt ggctgaggta gctgaagacg gccatccaca cctccctgtg
     5341 catgacgtgg gctgccccat cgtccagggg tagcgagtca ggcggggggc tgatgggggg
     5401 tggccggatc acatggcgct ccatctggat acacttgggc ggggacacgg agggtggggg
     5461 ccgggagatg acacggggcg ggctgcgcag gctgggcccc agctggtgcc gcagctcccg
     5521 gggggtgccg ttgagcccct tgctgaagcg gtggggacgc tcgcagatgc ccgggggccg
     5581 cttgggctcc tcgccctcgc tctcaggctc cgacttgatg ggctgctggt tctcgttggc
     5641 caggctgttc tccgtcctct ggatctcgtg gttgagctcc ttgctcagct ccctgctcag
     5701 ctccttgttg ggaagccgcc gcttccggcg catcttcacc ttcttcttct cctccgggcc
     5761 ctcggcccct tcggtgctgg gtcccgcggt gggggagctg gagcgggagt ggtcgctctc
     5821 cctggtcttg gggggcgcct cgggcagttc gtcctcgggt tcctgcttga agcgccggag
     5881 gggcttgttg gccagcgcca tgcggtcctc ggcgttcttc caggaccgcc gctttttcct
     5941 gaaaagctta tcttctttgc caggtttgag ctgctgctgg aagtaagtga gactggatct
     6001 ccaccagggg ctgaggctgc tgcctagagg gggcctcggc gagaggtgag aggaggaacc
     6061 gggggacgtt tgaagcgatg aggcccgctt gcgtccactc agctcctggg gcttctcgta
     6121 tttccgcttc ttcctcaggt gcacgtcgtc agactttctg cgcagaaggc cgtccggcgg
     6181 caccttcttc gagtgctcat ccgacctgcg ccggggcgcc tcctcacact cactcctccg
     6241 cttggcaggt tcctgccctg tggagagaaa ggcaaagtgg atgtcagtaa gaccaatagg
     6301 tgcctatcag aaacgcaaga gtcttctctg tctcgacaag cccagtttca taaattgcta
     6361 taaagtatcc tatacgaagt tatactagtt attggtctcc ttaaacctgt cttgtaacct
     6421 tgatacttac ctaatcgcca tcttccagca ggcgcaccat tgcccctgtt tcactatcca
     6481 ggttacggat atagttca
//
