LOCUS       sequence-479486-        7011 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for NEO1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7011)
  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  JOURNAL   Unpublished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 7011)
  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            "Unpublished"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' NEO1
FEATURES             Location/Qualifiers
     source          1..7011
                     /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"
     CDS             3731..3796
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3861..3881)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4274..4414
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4274..4403
                     /label=AAV2 ITR
     rep_origin      4489..4944
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     4786..4807
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4961..4980)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5080..5102
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5140..5158)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5226..5330
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5331..5399,5400..6191)
                     /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(5549..5568)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6362..6950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6851..6870
                     /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 ggtcttgatc gggggactga
     1501 gtataatttc cgagtggctg ctctaacaat caatggtaca ggcccggcaa ctgactggct
     1561 gtctgctgaa acttttgaaa gtgacctaga tgaaactcgt gttcctgaag tgcctagctc
     1621 tcttcacgta cgcccgctcg ttactagcat cgtagtgagc tggactcctc cagagaatca
     1681 gaacattgtg gtcagaggtt acgccattgg ttatggcatt ggcagccctc atgcccagac
     1741 catcaaagtg gactataaac agcgctatta caccattgaa aatctggatc ccagctctca
     1801 ctatgtgatt accctgaaag catttaataa cgtgggtgaa ggcatccccc tgtatgagag
     1861 tgctgtgacc aggcctcaca cagtgccaga tcccactccc atgatgccac cagtgggagt
     1921 tcaggcttcc attctgagtc atgacaccat caggattacg tgggcagaca actcgctgcc
     1981 caagcaccag aagattacag actcccgata ctacaccgtc cgatggaaaa ccaacatccc
     2041 agcaaacacc aagtacaaga atgcaaatgc aaccactttg agttatttgg tgactggttt
     2101 aaagccgaat acactctatg aattctctgt gatggtgacc aaaggtcgaa gatcaagtac
     2161 atggagtatg acagcccatg ggaccacctt tgaattagtt ccgacttctc cacccaagga
     2221 tgtgactgtt gtgagtaaag aggggaaacc taagaccata attgtgaatt ggcagcctcc
     2281 ctccgaagcc aatggcaaaa ttacaggtta catcatatat tacagtacag atgtgaatgc
     2341 agagatacat gactgggtta ttgagcctgt tgtgggaaac agactgactc accagataca
     2401 agagttaact cttgacacac catactactt caaaatccag gcacggaact caaagggcat
     2461 gggacccatg tctgaagctg tccaattcag aacacctaaa gcggactcct ctgataaaat
     2521 gcctaatgat caagcctcag ggtctggagg gaaaggaagc cggctgccag acctaggatc
     2581 cgactacaaa cctccaatga gcggcagtaa cagccctcat gggagcccca cctctcctct
     2641 ggacagtaat atgctgctgg tcataattgt ttctgttggc gtcatcacca tcgtggtggt
     2701 tgtgattatc gctgtctttt gtacccgtcg taccacctct caccagaaaa agaaacgagc
     2761 tgcctgcaaa tcagtgaatg gctctcataa gtacaaaggg aattccaaag atgtgaaacc
     2821 tccagatctc tggatccatc atgagagact ggagctgaaa cccattgata agtctccaga
     2881 cccaaacccc atcatgactg atactccaat tcctcgcaac tctcaagata tcacaccagt
     2941 tgacaactcc atggacagca atatccatca aaggcgaaat tcatacagag ggcatgagtc
     3001 agaggacagc atgtctacac tggctggaag gcgaggaatg agaccaaaaa tgatgatgcc
     3061 ctttgactcc cagccacccc agcctgtgat tagtgcccat cccatccatt ccctcgataa
     3121 ccctcaccat catttccact ccagcagcct cgcttctcca gctcgcagtc atctctacca
     3181 cccgggcagc ccatggccca ttggcacatc catgtccctt tcagacaggg ccaattccac
     3241 agaatccgtt cgaaataccc ccagcactga caccatgcca gcctcttcgt ctcaaacatg
     3301 ctgcactgat caccaggacc ctgaaggtgc taccagctcc tcttacttgg ccagctccca
     3361 agaggaagat tcaggccaga gtcttcccac tgcccatgtt cgcccttccc acccattgaa
     3421 gagcttcgcc gtgccagcaa tcccgcctcc aggacctccc acctatgatc ctgcattgcc
     3481 aagcacacca ttactgtccc agcaagctct gaaccatcac attcactcag tgaagacagc
     3541 ctccatcggg actctaggaa ggagccggcc tcctatgcca gtggttgttc ccagtgcccc
     3601 tgaagtgcag gagaccacaa ggatgttgga agactccgag agtagctatg aaccagatga
     3661 gctgaccaaa gagatggccc acctggaagg actaatgaag gacctaaacg ctatcacaac
     3721 agcatgtaca gactacaaag accatgacgg tgattataaa gatcatgaca tcgactacaa
     3781 ggatgacgat gacaagtaac tcgagataat caacctctgg attacaaaat ttgtgaaaga
     3841 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3901 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3961 tggttagttc ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     4021 gctcggctgt tgggcactga caattccgtg gtgtttattt gtgaaatttg tgatgctatt
     4081 gctttatttg taaccatcta gctttatttg tgaaatttgt gatgctattg ctttatttgt
     4141 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     4201 ggttcagggg gagatgtggg aggtttttta aagatctgga gaccggtgtg gcacgtgcgg
     4261 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4321 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4381 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4441 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4501 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4561 gcgccttagc gcccgctcct ttcgctttct ccccttcctt tctcgccacg ttcgccggct
     4621 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4681 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4741 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4801 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     4861 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4921 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     4981 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5041 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5101 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5161 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5221 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5281 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5341 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5401 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5461 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5521 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5581 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5641 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5701 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5761 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5821 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5881 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5941 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6001 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6061 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6121 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6181 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6241 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6301 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6361 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6421 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6481 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6541 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6601 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6661 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6721 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6781 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6841 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6901 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6961 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
