LOCUS       sequence-491875-        6759 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  3' AAVLINK plasmid for CLASP1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6759)
  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  JOURNAL   Unpublished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 6759)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 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            "Unpublished"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'-CLASP1-FLAG
FEATURES             Location/Qualifiers
     source          1..6759
                     /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"
     RBS             3058..3066
                     /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)"
     CDS             3479..3544
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3609..3629)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4022..4162
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4022..4151
                     /label=AAV2 ITR
     rep_origin      4237..4692
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4324..4343)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4534..4555
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4709..4728)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4828..4850
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4888..4906)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4974..5078
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5079..5147,5148..5939)
                     /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(5297..5316)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6110..6698
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6599..6618
                     /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 gggcttctgg gcctgcagaa
     1501 cttactgaag agccaaagaa cactgagtcg agttgaactg aaaaggttgt gtgagatctt
     1561 cactcggatg tttgctgacc ctcatagcaa gagagttttc agtatgtttt tggagactct
     1621 tgtggatttt ataataattc ataaggatga tttacaagac tggctttttg ttcttctcac
     1681 acaattactt aagaaaatgg gagcagattt acttggatct gtgcaagcaa aagttcaaaa
     1741 ggctctagat gtcacaaggg actcctttcc atttgatcaa caatttaaca ttttgatgag
     1801 atttattgtg gatcaaactc aaactccaaa cctcaaggtc aaagttgcaa tcctgaaata
     1861 cattgagtct ctggccagac agatggatcc aacagatttt gtaaactcta gtgagacaag
     1921 gcttgctgtt tctagaatca taacctggac aacagaacca aagagttcag acgtgagaaa
     1981 ggcagcacag attgtgctaa tctctctgtt tgaattgaat actcctgaat ttaccatgtt
     2041 acttggtgcc ttgccaaaaa cattccagga tggtgccacc aaactcctgc acaaccacct
     2101 caagaattcc agtaacacca gtgtgggctc tccaagcaat acgattggcc ggacgccctc
     2161 ccgacacacc agcagcagga ccagccccct gacctcaccc accaactgtt cccatggggg
     2221 tctgtctcca agtcggttat ggggttggag tgccgacggg ttagcgaagc acccacctcc
     2281 cttttctcag cctaactcca tccccaccgc tccctcccac aaggctctca ggcgctctta
     2341 ctctcccagc atgctggact atgatacaga gaacctgaac tctgaagaaa tctatagttc
     2401 tctacgtgga gttacagaag ccattgaaaa gtttagtttt cgaagccaag aagatctgaa
     2461 tgagccaatt aaacgagatg gcaaaaagga gtgtgatatt gtgtcccgcg atgggggcgc
     2521 tgcctcccct gccactgagg gccggggggg tagtgaagta gaaggaggcc ggacagctct
     2581 ggataacaag acctcactac tcaacaccca gcctccgcgc gccttcccgg ggccgcgggc
     2641 gcgagactac aacccgtacc cctactcaga tgccatcaac acctacgaca agaccgccct
     2701 gaaagaggct gtgttcgatg acgacatgga gcagcttcga gacgtgccca tcgaccattc
     2761 tgacctggtg gctgaccttc tgaaagagct gtccaaccac aatgagcgag tggaggaacg
     2821 gaagggagcc ctgctggagc tgctcaagat cacgcgggaa gacagccttg gtgtctggga
     2881 ggagcacttc aagaccattc tgctcctgct gctggagacc cttggagaca aagaccattc
     2941 aattcgagca ctggcgttaa gagttttgag ggaaattctg agaaatcaac cagcaagatt
     3001 taaaaactac gccgagctga cgattatgaa gactctggaa gcccacaaag actcccataa
     3061 ggaggtggtg agagcggctg aggaggctgc gtccacactg gccagttcca tccacccgga
     3121 gcagtgcatc aaggtgctct gccccatcat ccagacggcc gactacccca tcaaccttgc
     3181 tgccatcaag atgcagacca aagtcgtcga gaggatcgca aaggagtcat tgctgcagct
     3241 ccttgtcgac atcatcccag gcttgctgca gggttatgac aacaccgaaa gtagtgtgcg
     3301 taaggccagc gtgttttgct tagtggcaat ttattccgta atcggagaag acctgaaacc
     3361 tcaccttgca cagctcacag ggagcaagat gaagctacta aacttataca taaagagggc
     3421 ccagaccacc aacagcaaca gcagctcctc ctccgatgtc tccacgcaca gctgtacaga
     3481 ctacaaagac catgacggtg attataaaga tcatgacatc gactacaagg atgacgatga
     3541 caagtaactc gagataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3601 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3661 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttagttctt
     3721 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3781 ggcactgaca attccgtggt gtttatttgt gaaatttgtg atgctattgc tttatttgta
     3841 accatctagc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3901 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     3961 gatgtgggag gttttttaaa gatctggaga ccggtgtggc acgtgcggac cgagcggccg
     4021 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     4081 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     4141 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     4201 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     4261 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     4321 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4381 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4441 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4501 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4561 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     4621 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4681 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4741 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4801 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4861 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4921 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4981 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5041 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5101 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5161 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5221 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5281 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5341 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5401 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5461 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5521 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5581 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5641 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5701 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5761 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     5821 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5881 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5941 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6001 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6061 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6121 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6181 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6241 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6301 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6361 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6421 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6481 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6541 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6601 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6661 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6721 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
