LOCUS       sequence-476866-        6756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for ROBO2 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6756)
  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  JOURNAL   Unpublished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 6756)
  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            "Unpublished"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' ROBO2
FEATURES             Location/Qualifiers
     source          1..6756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     96..115
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             2108..2173
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2238..2258)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2651..2791
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2651..2780
                     /label=AAV2 ITR
     rep_origin      2866..3321
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2953..2972)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3163..3184
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3338..3357)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3457..3479
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3517..3535)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3603..3707
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3708..3776,3777..4568)
                     /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(3926..3945)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4739..5327
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5228..5247
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5389..5518
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             5665..6696
                     /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"
ORIGIN
        1 ggatacttta tagcaattta tgaaactggg cttgtcgaga cagagaagac tcttgcgttt
       61 ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca caggtattca
      121 ataccgggta gaggttgcag ctagtaccag tgcaggggtt ggagtaaaga gtgagccaca
      181 gccaataata atcgggagac gcaatgaagt tgtcattact gaaaacaata acagcataac
      241 tgagcaaatc actgatgtgg tgaagcaacc agcctttata gctggtattg gtggtgcctg
      301 ctgggtaatt ctgatgggtt ttagcatatg gttgtattgg cgaagaaaga agaggaaggg
      361 actcagtaat tatgctgtta cgtttcaaag aggagatgga ggactaatga gcaatggaag
      421 ccgtccaggt cttctcaatg ctggtgatcc cagctatcca tggcttgctg attcttggcc
      481 agccacgagc ttgccagtaa ataatagcaa cagtggccca aatgagattg gaaattttgg
      541 ccgtggagat gtgctgccac cagttccagg ccaaggggat aaaacagcaa cgatgctctc
      601 agatggagcc atttatagta gcattgactt cactaccaaa accagttaca acagttccag
      661 ccaaataaca caggctaccc catatgccac gacacagatc ttgcattcca acagcataca
      721 tgaattggct gtcgatctgc ctgatccaca atggaaaagc tcaattcagc aaaaaacaga
      781 tctgatggga tttggttatt ctctacctga tcagaacaaa ggtaacaatg gtgggaaagg
      841 tggaaaaaag aagaaaaata aaaactcttc taaaccacag aaaaacaatg gatccacttg
      901 ggccaatgtc cctctacctc cccccccagt ccagcccctt cctggcacgg agctggaaca
      961 ctatgcagtg gaacaacaag aaaatggcta tgacagtgat agctggtgcc caccattgcc
     1021 agtacaaact tacttacacc aaggtctgga agatgaactg gaagaagatg atgatagggt
     1081 cccaacacct cctgttcgag gcgtggcttc ttctcctgct atctcctttg gacagcagtc
     1141 cactgcaact cttactccat ccccacggga agagatgcaa cccatgctgc aggctcacct
     1201 ggatgagttg acaagagcct atcagtttga tatagcaaaa caaacatggc acattcaaag
     1261 caataatcaa cctccacagc ctccagttcc accgttaggt tatgtgtctg gagccttgat
     1321 ttctgatttg gaaacggatg ttgcagatga tgatgccgac gacgaagagg aagctttaga
     1381 aatccccagg cccctgagag cactggacca gactcctgga tccagcatgg acaatctaga
     1441 cagctctgtg acaggaaaag cctttacctc ctctcaaaga cctcgaccta ccagcccatt
     1501 ttctactgac agtaacacca gtgcagccct gagtcaaagt cagaggcctc ggcccactaa
     1561 aaaacacaag ggagggcgga tggaccaaca accagcattg cctcatcgaa gggaaggaat
     1621 gacagatgat cttccaccac caccagatcc cccgccaggt cagggtttaa ggcagcaaat
     1681 aggcccgagc cagcaggctg gtaacgtgga aaactcagca gagagaaaag gaagctctct
     1741 agagagacaa catgcatcca gcttagaaga cacaaagagc tcattggatt gtccagctag
     1801 aacctcccta gagtggcagc gacaaaccca ggaatggata agctccacag aacgacaaga
     1861 agatatacgg aaagccccac acaaacaagg tgtcggatca gaggaggcct tggtgcccta
     1921 tagcaagccc agtttcccat ctccaggtgg ccacagctca tcaggaacag cttcttctaa
     1981 gggatccact ggacctagga aaaccgaggt gttgagagca ggccaccagc gcaatgccag
     2041 cgaccttctt gacataggat atatgggctc caacagtcaa ggacagttta caggtgaatt
     2101 atgtacagac tacaaagacc atgacggtga ttataaagat catgacatcg actacaagga
     2161 tgacgatgac aagtaactcg agataatcaa cctctggatt acaaaatttg tgaaagattg
     2221 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     2281 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     2341 ttagttcttg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     2401 cggctgttgg gcactgacaa ttccgtggtg tttatttgtg aaatttgtga tgctattgct
     2461 ttatttgtaa ccatctagct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     2521 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     2581 tcagggggag atgtgggagg ttttttaaag atctggagac cggtgtggca cgtgcggacc
     2641 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     2701 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     2761 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     2821 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     2881 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2941 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3001 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3061 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     3121 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3181 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     3241 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3301 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     3361 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3421 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3481 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     3541 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     3601 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3661 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3721 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3781 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     3841 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3901 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     3961 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4021 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4081 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4141 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4201 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4261 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4321 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4381 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     4441 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     4501 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4561 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4621 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4681 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4741 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4801 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4861 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4921 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4981 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5041 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5101 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5161 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5221 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5281 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5341 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc
     5401 tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc
     5461 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc
     5521 ggccgcacgc gtcgctctgc gcgaggcggc tccgccgcgg cggagggata cggcgcacca
     5581 tatatatatc gcggggcgca gactcgcgct ccggcagtgg tgctgggagt gtcgtggacg
     5641 ccgtgccgtt actcgtacgc caccatgtcc aatttactga ccgtacacca aaatttgcct
     5701 gcattaccgg tcgatgcaac gagtgatgag gttcgcaaga acctgatgga catgttcagg
     5761 gatcgccagg cgttttctga gcatacctgg aaaatgcttc tgtccgtttg ccggtcgtgg
     5821 gcggcatggt gcaagttgaa taaccggaaa tggtttcccg cagaacctga agatgttcgc
     5881 gattatcttc tatatcttca ggcgcgcggt ctggcagtaa aaaccatcca gcaacatttg
     5941 ggccagctaa acatgcttca tcgtcggtcc gggctgccac gaccaagtga cagcaatgct
     6001 gtttcactgg ttatgcggcg gatccgaaaa gaaaacgttg atgccggtga acgtgcaaaa
     6061 caggctctag cgttcgaacg cactgatttc gaccaggttc gttcactcat ggaaaatagc
     6121 gatcgctgcc aggatatacg taatctggca tttctgggga ttgcttataa caccctgtta
     6181 cgtatagccg aaattgccag gatcagggtt aaagatatct cacgtactga cggtgggaga
     6241 atgttaatcc atattggcag aacgaaaacg ctggttagca ccgcaggtgt agagaaggca
     6301 cttagcctgg gggtaactaa actggtcgag cgatggattt ccgtctctgg tgtagctgat
     6361 gatccgaata actacctgtt ttgccgggtc agaaaaaatg gtgttgccgc gccatctgcc
     6421 accagccagc tatcaactcg cgccctggaa gggatttttg aagcaactca tcgattgatt
     6481 tacggcgcta aggatgactc tggtcagaga tacctggcct ggtctggaca cagtgcccgt
     6541 gtcggagccg cgcgagatat ggcccgcgct ggagtttcaa taccggagat catgcaagct
     6601 ggtggctgga ccaatgtaaa tattgtaatg aactatatcc gtaacctgga tagtgaaaca
     6661 ggggcaatgg tgcgcctgct ggaagatggc gattaggtaa gtatcaaggt tacaagacag
     6721 gtttaaggag accaataact agtataactt cgtata
//
