LOCUS       sequence-491911-        6652 bp    DNA     circular SYN 05-NOV-2025
DEFINITION  3' AAVLINK plasmid for KIF14 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6652)
  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  JOURNAL   Unpublished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 6652)
  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            "Unpublished"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'-KIF14-FLAG
FEATURES             Location/Qualifiers
     source          1..6652
                     /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             3372..3437
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3502..3522)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3915..4055
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3915..4044
                     /label=AAV2 ITR
     rep_origin      4130..4585
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4217..4236)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4427..4448
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4602..4621)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4721..4743
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4781..4799)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4867..4971
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4972..5040,5041..5832)
                     /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(5190..5209)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6003..6591
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6492..6511
                     /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 ggaaatatat gtcaacaaaa
     1501 agcgattaga aatggagaca ttggctacaa aacaggcttt agaagaccat agcatccgcc
     1561 atgcaagaat tctggaagct ttagaaactg aaaagcaaaa aattgctaaa gaagtacaaa
     1621 ttctacagca gaatcggaat aatagggata aaacttttac agtgcagaca acttggagct
     1681 ctatgaaact ctcaatgatg attcaggaag ccaatgctat cagcagcaaa ttgaaaacat
     1741 actatgtttt tggcagacat gatatatcag ataaaagtag ttctgacact tctattcggg
     1801 ttcgtaacct gaaactagga atctcaacat tctggagtct ggaaaagttt gaatctaaac
     1861 ttgcagcaat gaaagaactt tatgagagta atggtagtaa caggggtgaa gatgcctttt
     1921 gtgatcctga agatgaatgg gaacccgaca ttacagatgc accagtttct tcactttcta
     1981 gaaggaggag taggagtttg atgaagaaca gaagaatttc tggttgttta catgacatac
     2041 aagtccatcc aattaagaat ttgcattctt cacattcatc aggtttaatg gacaaatcaa
     2101 gcactattta ctcaaattca gcagagtcct ttcttcctgg aatttgcaaa gaattgattg
     2161 gttcttcgtt agattttttt ggacagagtt atgatgaaga aagaactata gcagacagcc
     2221 taattaatag ttttcttaaa atttataatg ggctatttgc catttccaag gctcatgaag
     2281 aacaagatga agaaagtcaa gataacttgt tttcttctga tcgagcaatc cagtcactta
     2341 ctattcagac tgcatgtgct tttgagcagc tagtagtgct aatgaaacac tggctgagtg
     2401 atttactgcc ttgtaccaac atagcaagac ttgaggatga attgagacaa gaagttaaaa
     2461 aactgggagg ctacttacag ttatttttgc agggatgctg tttggatatt tcatcaatga
     2521 taaaagaggc tcaaaagaat gcaatccaaa ttgtacaaca agctgtaaag tatgtggggc
     2581 agttagcagt tctgaaaggg agcaagctac attttctaga aaacggtaac aataaagctg
     2641 ccagtgtcca ggaggaattc atggatgctg tttgtgatgg tgtaggctta ggaatgaaga
     2701 ttttattaga ttctggactg gaaaaagcaa aagaacttca gcatgaactc tttaggcagt
     2761 gtacaaaaaa tgaggttacc aaagaaatga aaactaatgc catgggattg attagatctc
     2821 ttgaaaacat ctttgctgaa tcgaaaatta aaagtttcag aaggcaagta caagaagaaa
     2881 actttgaata ccaagatttc aagaggatgg ttaatcgtgc tccagaattc ttaaagttaa
     2941 aacattgctt agagaaagct attgaaatta ttatttctgc actgaaagga tgccatagtg
     3001 atataaatct tctccagact tgtgttgaaa gtattcgcaa cttggccagt gatttttaca
     3061 gtgacttcag tgtgccttct acttctgttg gcagctatga gagtagagta actcacattg
     3121 tccaccagga actagaatct ctagctaagt ctctcctctt ttgttttgaa tctgaagaaa
     3181 gccctgattt gttgaaaccc tgggaaactt ataatcaaaa taccaaagaa gaacaccaac
     3241 aatctaaatc aagcgggatt gacggcagta agaataaagg tgtaccaaag cgtgtctatg
     3301 agctccatgg ctcatcccca gcagtgagct cagaggaatg cacacccagt aggattcagt
     3361 gggtgtgtac agactacaaa gaccatgacg gtgattataa agatcatgac atcgactaca
     3421 aggatgacga tgacaagtaa ctcgagataa tcaacctctg gattacaaaa tttgtgaaag
     3481 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3541 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3601 ctggttagtt cttgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3661 ggctcggctg ttgggcactg acaattccgt ggtgtttatt tgtgaaattt gtgatgctat
     3721 tgctttattt gtaaccatct agctttattt gtgaaatttg tgatgctatt gctttatttg
     3781 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     3841 aggttcaggg ggagatgtgg gaggtttttt aaagatctgg agaccggtgt ggcacgtgcg
     3901 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     3961 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4021 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4081 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4141 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4201 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4261 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4321 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4381 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4441 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     4501 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4561 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4621 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4681 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4741 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4801 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4861 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4921 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4981 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5041 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5101 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5161 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5221 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5281 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5341 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5401 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5461 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5521 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5581 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5641 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5701 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5761 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5821 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5881 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5941 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6001 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6061 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6121 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6181 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6241 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6301 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6361 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6421 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6481 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6541 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6601 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
