LOCUS       sequence-491436-        7027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3' AAVLINK plasmid for TCF20 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7027)
  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  JOURNAL   Unpublished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 7027)
  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            "Unpublished"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'-TCF20-FLAG
FEATURES             Location/Qualifiers
     source          1..7027
                     /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             3736..3801
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3878..3898)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4290..4430
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4290..4419
                     /label=AAV2 ITR
     rep_origin      4505..4960
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4592..4611)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4802..4823
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4977..4996)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5096..5118
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5156..5174)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5242..5346
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5347..5415,5416..6207)
                     /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(5565..5584)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6378..6966
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6867..6886
                     /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 ggtatgggcc gccccatgag
     1501 actgatggac atggactagc tgaggctaca cagtcatcca aacctggtag tgttatgctg
     1561 agacttccag gccaggagga tcattcttct caaaacccct taatcatgag gaggcgtgtt
     1621 cgttctttta tctctcccat tcccagtaag agacagtcac aagatgtaaa gaacagtagc
     1681 actgaagata aaggtcgcct ccttcactca tcaaaagaag gcgctgataa agcattcaat
     1741 tcctatgccc atctttctca cagtcaggat atcaagtcta tccctaagag agattcctcc
     1801 aaggaccttc caagtccaga tagtagaaac tgccctgctg ttaccctcac aagccctgct
     1861 aagaccaaaa tactgccccc acggaaagga cggggattga aattggaagc tatagttcag
     1921 aagattacat ccccaaatat taggaggagc gcatcttcga acagtgcgga ggctggggga
     1981 gacacggtta cgcttgatga tatactgtct ttgaagagtg gtcctcctga aggtgggagt
     2041 gttgctgttc aggatgctga catagagaag agaaaaggtg aggtggcttc ggacctagtc
     2101 agtccagcaa accaggagtt gcacgtagag aaacctcttc caaggtcttc agaagagtgg
     2161 cgtggcagcg tggatgacaa agtgaagaca gagacacatg cagaaacagt tactgccgga
     2221 aaggaacccc ctggtgccat gacatccaca acctcacaga agcctggtag taaccaaggg
     2281 agaccagatg gttccctggg tggaacagca cctttaatct ttccagactc aaagaatgta
     2341 cctccagtgg gcatattggc ccctgaggca aaccccaagg ctgaagagaa ggagaacgat
     2401 acagtgacga tttcaccgaa gcaagagggt ttccctccaa agggatattt cccatcagga
     2461 aagaagaagg ggagacccat tggtagtgtg aataagcaaa agaaacagca gcagccaccg
     2521 cctccacccc ctcagccccc acagatacca gaaggttctg cagatggaga gccaaagcca
     2581 aaaaaacaga ggcaaaggag ggagagaagg aagcctgggg cccagccgag gaagcgaaaa
     2641 accaaacaag cagttcccat tgtggaaccc caagaacctg agatcaaact aaaatatgcc
     2701 acccagccac tggataaaac tgatgccaag aacaagtctt tttaccctta catccatgta
     2761 gtaaataagt gtgaacttgg agccgtttgt acaatcatca atgctgagga agaagaacag
     2821 accaaattag tgaggggcag gaagggtcag aggtcactga cccctccacc tagcagcact
     2881 gaaagcaagg cgctcccggc ctcgtccttt atgctgcagg gacctgttgt gacagagtct
     2941 tcggttatgg ggcacctggt ttgctgtctg tgtggcaagt gggccagtta ccggaacatg
     3001 ggtgacctct ttggaccttt ttatccccaa gattatgcag ccactctccc gaagaatcca
     3061 cctcctaaga gggccacaga aatgcagagc aaagttaagg tacggcacaa aagtgcttct
     3121 aatggctcca agacggacac tgaggaggag gaagagcagc agcagcagca gaaggagcag
     3181 agaagcctgg ccgcacaccc caggtttaag cggcgccacc gctcggaaga ctgtggtgga
     3241 ggccctcggt ccctgtccag ggggctccct tgtaaaaaag cagccactga gggcagcagt
     3301 gaaaagactg ttttggactc gaagccctcc gtgcccacca cttcagaagg tggccctgag
     3361 ctggagttac aaatccctga actacctctt gacagcaatg aattttgggt ccatgagggt
     3421 tgtattctct gggccaatgg aatctacctg gtttgtggca ggctctatgg cctgcaggaa
     3481 gcgctggaaa tagccagaga gatgaaatgt tcccactgcc aggaggcagg cgccaccttg
     3541 ggctgctaca acaaaggctg ctccttccga taccattacc cgtgtgccat tgatgcagat
     3601 tgtttgctac atgaggagaa cttctcggtg aggtgcccta agcacaagcc tccccttccg
     3661 tgccctctcc cccccttgca gaacaagacc gcgaaaggca gcctcagcac agagcagtcg
     3721 gagcgggggt gtacagacta caaagaccat gacggtgatt ataaagatca tgacatcgac
     3781 tacaaggatg acgatgacaa gtaagaattc tgatgactcg agataatcaa cctctggatt
     3841 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3901 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3961 cctccttgta taaatcctgg ttagttcttg ccacggcgga actcatcgcc gcctgccttg
     4021 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg tttatttgtg
     4081 aaatttgtga tgctattgct ttatttgtaa ccatctagct ttatttgtga aatttgtgat
     4141 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     4201 attcatttta tgttcaggtt cagggggaga tgtgggaggt tttttaaaga tctggagacc
     4261 ggtgtggcac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc
     4321 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4381 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     4441 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     4501 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4561 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     4621 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4681 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     4741 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4801 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     4861 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     4921 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     4981 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5041 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5101 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5161 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5221 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5281 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5341 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5401 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5461 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5521 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5581 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5641 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5701 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5761 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5821 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5881 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5941 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6001 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6061 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6121 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6181 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6241 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6301 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6361 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6421 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6481 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6541 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6601 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6661 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6721 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6781 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6841 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6901 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6961 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7021 cacatgt
//
