LOCUS       sequence-491392-        5739 bp    DNA     circular SYN 03-NOV-2025
DEFINITION  3' AAVLINK plasmid for RALGAPB expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5739)
  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  JOURNAL   Unpublished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 5739)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 3, 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            "Unpublished"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'-RALGAPB-FLAG
FEATURES             Location/Qualifiers
     source          1..5739
                     /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             2459..2524
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2589..2609)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3002..3142
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3002..3131
                     /label=AAV2 ITR
     rep_origin      3217..3672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3304..3323)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3514..3535
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3689..3708)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3808..3830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3868..3886)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3954..4058
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4059..4127,4128..4919)
                     /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(4277..4296)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5090..5678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5579..5598
                     /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 ggtcagaaaa cgaaccaaga
     1501 gattttaaag aatgtggagt cttccagaac tgttcagcca catttcctag aatttttgct
     1561 ttcccttggc tggtcagtag atgtgggcag acaccctggt tggactgggc atgtttctac
     1621 cagttggtct attaattgtt gtgatgatgg tgaaggatct caacaagaag tgatttcctc
     1681 tgaagatatt ggagctagca ttttcaatgg acagaagaag gtgctgtatt atgctgatgc
     1741 ccttacagaa attgcttttg tggttccttc tcctgtggag tccttaactg attcattgga
     1801 aagtaacatc tcggaccaag atagtgattc aaatatggat cttatgccag gaattctgaa
     1861 acagccatcc ctgacacttg agcttttccc caatcataca gacaatctta attcctcaca
     1921 gaggctcagt cccagttcca gaatgaggaa gctgcctcag ggtcgccctg ttcctcccct
     1981 tggacctgag acaagagttt ctgtagtctg ggtggaacgc tatgatgata tagaaaactt
     2041 tcccctctca gagctgatga cagagatcag tactggtgtg gaaactactg caaatagtag
     2101 cacttcactg agatctacaa ctcttgaaaa agaagttcct gtcatcttca tccacccttt
     2161 aaacactgga ttattccgga taaaaattca aggagccact ggaaaattta atatggtcat
     2221 ccctcttgtg gatgggatga ttgtcagcag gcgagctctt ggctttctgg tgaggcagac
     2281 tgtaattaac atttgtagaa gaaagagact ggaaagtgac tcctacagtc ccccccatgt
     2341 ccgccggaaa cagaaaatca ccgacattgt caacaagtac cggaacaagc agctggagcc
     2401 agagttttat acttcacttt tccaggaggt tggactcaag aactgcagtt cttgtacaga
     2461 ctacaaagac catgacggtg attataaaga tcatgacatc gactacaagg atgacgatga
     2521 caagtaactc gagataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     2581 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     2641 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttagttctt
     2701 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     2761 ggcactgaca attccgtggt gtttatttgt gaaatttgtg atgctattgc tttatttgta
     2821 accatctagc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     2881 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     2941 gatgtgggag gttttttaaa gatctggaga ccggtgtggc acgtgcggac cgagcggccg
     3001 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     3061 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     3121 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3181 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3241 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     3301 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3361 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3421 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3481 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3541 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     3601 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3661 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3721 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3781 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3841 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3901 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3961 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4021 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4081 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4141 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4201 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4261 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4321 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4381 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4441 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4501 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4561 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4621 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4681 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4741 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4801 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4861 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4921 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4981 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5041 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5101 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5161 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5221 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5281 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5341 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5401 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5461 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5521 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5581 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5641 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5701 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
