LOCUS       sequence-491934-        7047 bp    DNA     circular SYN 05-NOV-2025
DEFINITION  3' AAVLINK plasmid for CHD1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7047)
  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  JOURNAL   Unpublished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 7047)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 5, 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            "Unpublished"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'-CHD1-FLAG
FEATURES             Location/Qualifiers
     source          1..7047
                     /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"
     protein_bind    1712..1723
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     RBS             2617..2628
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3767..3832
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3897..3917)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4310..4450
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4310..4439
                     /label=AAV2 ITR
     rep_origin      4525..4980
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4612..4631)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4822..4843
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4997..5016)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5116..5138
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5176..5194)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5262..5366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5367..5435,5436..6227)
                     /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(5585..5604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6398..6986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6887..6906
                     /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 ggttctgccc catcaagttc
     1501 tactcctttc aataaagaag agttatcagc cattttaaag tttggtgctg aagaactttt
     1561 taaggaacct gaaggagaag aacaagagcc ccaggaaatg gatatagatg aaatcttgaa
     1621 gagagctgaa actcatgaaa atgaaccagg tcctttaact gtaggagatg aattgctttc
     1681 ccagttcaag gttgccaact tctcaaatat ggatgaggat gacattgagt tggaacctga
     1741 aagaaattca aagaattggg aggaaattat tccagaagat caaagaagac gattagaaga
     1801 agaagaaaga caaaaggaac ttgaagaaat ttatatgctc ccaagaatga gaaattgtgc
     1861 aaaacagatt agtttcaatg gaagtgaagg gaggcgcagt agaagtagga gatactctgg
     1921 atctgatagt gattccatct cagaagggaa aaggccaaag aaacgtggaa gaccacggac
     1981 tattcctcgg gagaatatta aaggatttag tgatgcagaa attaggcggt ttatcaagag
     2041 ctataagaaa tttggtggtc ctctggaaag attagatgca attgctcgag atgctgagtt
     2101 agttgataag tcagaaacag accttagacg actgggagaa ttggtacata atggttgcat
     2161 taaagcatta aaggatagtt cttcaggaac agaacgaaca ggtggtagac tcggaaaagt
     2221 gaagggtcca acattccgaa tatcaggagt acaggtgaat gccaaactag tcatctccca
     2281 tgaagaagaa ttaatacctt tgcacaaatc cattccttct gatccagaag aaagaaagca
     2341 gtatactatc ccatgccaca caaaggcagc tcattttgat atagactggg gcaaagaaga
     2401 tgattccaat ttgttaattg gcatctatga atatggatat ggaagctggg aaatgattaa
     2461 aatggatcct gacctcagtc taacacacaa gattcttcca gatgatcccg ataaaaaacc
     2521 acaagcaaaa cagttgcaga cccgtgcaga ctacctcatc aaattactta gtagagatct
     2581 tgcaaaaaaa gaagctcttt ctggtgcggg aagttcaaag aggagaaaag caagagctaa
     2641 gaagaataaa gcaatgaagt ctataaaagt gaaagaggaa ataaagagtg attcttctcc
     2701 tctgccttca gagaagtctg atgaagatga tgataaattg agtgaatcca agtctgatgg
     2761 tagggaaaga tccaagaaat cttcagtgtc agatgctcca gttcatatca cggcaagtgg
     2821 tgaaccagtt cccatttctg aagaatctga agagctggat cagaagacat tcagcatttg
     2881 taaagaaaga atgaggcctg ttaaagcagc tttgaaacaa cttgataggc ctgagaaagg
     2941 cctttcagaa agagaacaac tagagcatac tagacaatgt ttaataaaaa ttggagacca
     3001 tatcacagaa tgtctaaaag agtatacaaa tcctgaacaa attaagcaat ggagaaaaaa
     3061 cctgtggatt tttgtatcta agtttactga atttgatgca agaaaattac ataaattata
     3121 taagcatgct attaaaaaac ggcaggagtc tcagcaaaac agtgatcaaa acagcaactt
     3181 gaatcctcac gtgattagaa atccagatgt ggaaagatta aaagagaata caaatcacga
     3241 tgatagcagc agggacagtt attcctctga tagacactta actcagtacc atgatcatca
     3301 taaagaccga catcagggag attcttacaa aaaaagtgat tccaggaaaa gaccctattc
     3361 ttcttttagt aatggtaaag accatcgtga ttgggatcac tacaagcaag acagcagata
     3421 ttacagtgac agagagaaac acagaaaact ggatgatcac aggagtagag atcacaggtc
     3481 aaatttggaa ggaagtttaa aagatagatc tcattctgat catcgttctc actcagatca
     3541 tcggttacat tcagaccacc ggtcaagttc tgaatatacg caccataaat cttccaggga
     3601 ttataggtat cactcagact ggcaaatgga ccacagagct tccagcagtg gccctaggtc
     3661 accactagat cagagatctc cttatggctc cagatctcca tttgaacatt cagttgaaca
     3721 caaaagtaca ccggagcata cctggagtag tcggaaaaca tgtacagact acaaagacca
     3781 tgacggtgat tataaagatc atgacatcga ctacaaggat gacgatgaca agtaactcga
     3841 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     3901 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     3961 cgtatggctt tcattttctc ctccttgtat aaatcctggt tagttcttgc cacggcggaa
     4021 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     4081 tccgtggtgt ttatttgtga aatttgtgat gctattgctt tatttgtaac catctagctt
     4141 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     4201 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt
     4261 tttttaaaga tctggagacc ggtgtggcac gtgcggaccg agcggccgca ggaaccccta
     4321 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4381 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4441 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4501 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4561 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     4621 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4681 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4741 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4801 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     4861 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     4921 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     4981 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     5041 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5101 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5161 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5221 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5281 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5341 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5401 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5461 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5521 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5581 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5641 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5701 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5761 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5821 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5881 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5941 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6001 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6061 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6121 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6181 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6241 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6301 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6361 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6421 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6481 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6541 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6601 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6661 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6721 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6781 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6841 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6901 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6961 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7021 ccttttgctg gccttttgct cacatgt
//
