LOCUS       sequence-491929-        6337 bp    DNA     circular SYN 05-NOV-2025
DEFINITION  3' AAVLINK plasmid for KIF1A expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6337)
  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  JOURNAL   Unpublished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 6337)
  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            "Unpublished"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'-KIF1A-FLAG
FEATURES             Location/Qualifiers
     source          1..6337
                     /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"
     CDS             3045..3110
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3187..3207)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3600..3740
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3600..3729
                     /label=AAV2 ITR
     rep_origin      3815..4270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3902..3921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4112..4133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4287..4306)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4406..4428
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4466..4484)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4552..4656
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4657..4725,4726..5517)
                     /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(4875..4894)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5688..6276
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6177..6196
                     /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 gggcatccag cgacggatta
     1501 cggtgacact actgcatgag acaggcagcc atatccgctg gaaggaagtg cgcgagctgg
     1561 tcgtgggccg catccgaaac actccagaga ccgacgagtc cctgatcgac cccaacatct
     1621 tgtctctcaa catcctctct tccggataca tccacccagc ccaagatgac cggacctttt
     1681 accaatttga ggctgcgtgg gacagctcca tgcacaactc tctcctgctg aaccgggtca
     1741 ccccttatcg agagaaaatc tacatgacac tctccgctta tatcgagatg gagaactgca
     1801 cccagccggc tgttgtcacc aaggacttct gcatggtctt ctattcccgt gatgccaagc
     1861 tgccagcctc gcgctccatc cgcaacctct ttggcagtgg gagccttcgg gcctcagaga
     1921 gtaaccgtgt gactggtgtg tacgaactca gcctgtgcca cgtggctgac gcgggcagcc
     1981 cagggatgca gcgccggcgc cgacgagtcc tggacacatc tgtggcctat gtccggggcg
     2041 aggagaacct ggcaggctgg aggccccgga gtgacagtct cattctggac caccagtggg
     2101 agctggagaa gctgagcctc ctgcaggagg tggagaagac taggcactac ctgctcctgc
     2161 gggagaagct ggagaccgcc cagcggcctg tcccggaggt actgtccccg gccttcagcg
     2221 aggactctga gtcccatggc tcctccagcg cctcctcccc gctctcggct gagggccgcc
     2281 catcacccct ggaggctccc aacgagaggc agcgggagct ggccgtcaag tgcttgcgcc
     2341 tgctcacgca cacattcaac agagagtaca cacacagcca cgtctgcgtc agtgccagcg
     2401 agagcaagct ctccgagatg tctgtcaccc tgctccggga cccgtcgatg tcccctctag
     2461 gggtggccac tctcaccccc tcctccactt gcccctctct ggttgaaggg cggtacggtg
     2521 ccactgacct gaggaccccg cagccctgct cccggccagc cagcccagag cccgagctgc
     2581 tgccagaggc cgactccaag aagctccctt cccctgcccg ggcaacagag acagacaagg
     2641 agccccagcg cctgctggtc cctgacatcc aggagatccg agtcagcccg atcgtttcca
     2701 agaaggggta cctgcacttc ctggagccgc acacgtcagg ctgggccagg cgcttcgtgg
     2761 tggtgcggcg cccctatgcc tacatgtaca acagcgacaa ggacaccgtg gagcggttcg
     2821 tgctcaacct ggccactgcc caggtggagt acagtgagga ccagcaggct atgctcaaga
     2881 cacccaacac attcgcggtg tgcacggaac accgcggcat cctgctgcag gccgccagcg
     2941 acaaggacat gcatgactgg ctgtacgcct tcaaccccct cctggccggg accatacggt
     3001 ccaagctctc cagaaggagg tctgcccaga tgcgggtctg tacagactac aaagaccatg
     3061 acggtgatta taaagatcat gacatcgact acaaggatga cgatgacaag taagaattct
     3121 gatgactcga gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     3181 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     3241 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tagttcttgc
     3301 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     3361 cactgacaat tccgtggtgt ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3421 catctagctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     3481 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga
     3541 tgtgggaggt tttttaaaga tctggagacc ggtgtggcac gtgcggaccg agcggccgca
     3601 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     3661 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     3721 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3781 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     3841 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     3901 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3961 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4021 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4081 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4141 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     4201 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4261 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4321 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4381 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4441 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4501 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4561 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4621 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4681 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4741 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4801 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4861 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     4921 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4981 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5041 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5101 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5161 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5221 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5281 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5341 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5401 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5461 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5521 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5581 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5641 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5701 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5761 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5821 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5881 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5941 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6001 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6061 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6121 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6181 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6241 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6301 cggttcctgg ccttttgctg gccttttgct cacatgt
//
