LOCUS       sequence-491882-        7174 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  3' AAVLINK plasmid for TRPM1 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7174)
  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  JOURNAL   Unpublished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 7174)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 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            "Unpublished"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'-TRPM1-FLAG
FEATURES             Location/Qualifiers
     source          1..7174
                     /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             3894..3959
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4024..4044)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4437..4577
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4437..4566
                     /label=AAV2 ITR
     rep_origin      4652..5107
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4739..4758)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4949..4970
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5124..5143)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5243..5265
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5303..5321)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5389..5493
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5494..5562,5563..6354)
                     /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(5712..5731)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6525..7113
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7014..7033
                     /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 gggggatgag gagaacgagc
     1501 acaaaaaaca gagaagtatt cccatcggaa caaagatctg tgaattctat aacgcgccca
     1561 ttgtcaagtt ctggttttac acaatatcat acttgggcta cctgctgctg tttaactacg
     1621 tcatcctggt gcggatggat ggctggccgt ccctccagga gtggatcgtc atctcctaca
     1681 tcgtgagcct ggcgttagag aagatacgag agatcctcat gtcagaacca ggcaaactca
     1741 gccagaaaat caaagtttgg cttcaggagt actggaacat cacagatctc gtggccattt
     1801 ccacattcat gattggagca attcttcgcc tacagaacca gccctacatg ggctatggcc
     1861 gggtgatcta ctgtgtggat atcatcttct ggtacatccg tgtcctggac atctttggtg
     1921 tcaacaagta tctggggcca tacgtgatga tgattggaaa gatgatgatc gacatgctgt
     1981 actttgtggt catcatgctg gtcgtgctca tgagtttcgg agtagcccgt caagccattc
     2041 tgcatccaga ggagaagccc tcttggaaac tggcccgaaa catcttctac atgccctact
     2101 ggatgatcta tggagaggtg tttgcagacc agatagacct ctacgccatg gaaattaatc
     2161 ctccttgtgg tgagaaccta tatgatgagg agggcaagcg gcttcctccc tgtatccccg
     2221 gcgcctggct cactccagca ctcatggcgt gctatctact ggtcgccaac atcctgctgg
     2281 tgaacctgct gattgctgtg ttcaacaata ccttctttga agtaaaatca atatccaacc
     2341 aggtgtggaa gttccagcga tatcagctga ttatgacatt tcatgacagg ccagtcctgc
     2401 ccccaccgat gatcatttta agccacatct acatcatcat tatgcgtctc agcggccgct
     2461 gcaggaaaaa gagagaaggg gaccaagagg aacgggatcg tggattgaag ctcttcctta
     2521 gcgacgagga gctaaagagg ctgcatgagt tcgaggagca gtgcgtgcag gagcacttcc
     2581 gggagaagga ggatgagcag cagtcgtcca gcgacgagcg catccgggtc acttctgaaa
     2641 gagttgaaaa tatgtcaatg aggttggaag aaatcaatga aagagaaact tttatgaaaa
     2701 cttccctgca gactgttgac cttcgacttg ctcagctaga agaattatct aacagaatgg
     2761 tgaatgctct tgaaaatctt gcgggaatcg acaggtctga cctgatccag gcacggtccc
     2821 gggcttcttc tgaatgtgag gcaacgtatc ttctccggca aagcagcatc aatagcgctg
     2881 atggctacag cttgtatcga tatcatttta acggagaaga gttattattt gaggatacat
     2941 ctctctccac gtcaccaggg acaggagtca ggaaaaaaac ctgttccttc cgtataaagg
     3001 aagagaagga cgtgaaaacg cacctagtcc cagaatgtca gaacagtctt cacctttcac
     3061 tgggcacaag cacatcagca accccagatg gcagtcacct tgcagtagat gacttaaaga
     3121 acgctgaaga gtcaaaatta ggtccagata ttgggatttc aaaggaagat gatgaaagac
     3181 agacagactc taaaaaagaa gaaactattt ccccaagttt aaataaaaca gatgtgatac
     3241 atggacagga caaatcagat gttcaaaaca ctcagctaac agtggaaacg acaaatatag
     3301 aaggcactat ttcctatccc ctggaagaaa ccaaaattac acgctatttc cccgatgaaa
     3361 cgatcaatgc ttgtaaaaca atgaagtcca gaagcttcgt ctattcccgg ggaagaaagc
     3421 tggtcggtgg ggttaaccag gatgtagagt acagttcaat cacggaccag caattgacga
     3481 cggaatggca atgccaagtt caaaagatca cgcgctctca tagcacagat attccttaca
     3541 ttgtgtcgga agctgcagtg caagctgagc ataaagagca gtttgcagat atgcaagatg
     3601 aacaccatgt cgctgaagca attcctcgaa tccctcgctt gtccctaacc attactgaca
     3661 gaaatgggat ggaaaactta ctgtctgtga agccagatca aactttggga ttcccatctc
     3721 tcaggtcaaa aagtttacat ggacatccta ggaatgtgaa atccattcag ggaaagttag
     3781 acagatctgg acatgccagt agtgtaagca gcttagtaat tgtgtctgga atgacagcag
     3841 aagaaaaaaa ggttaagaaa gagaaagctt ccacagaaac tgaatgctgt acagactaca
     3901 aagaccatga cggtgattat aaagatcatg acatcgacta caaggatgac gatgacaagt
     3961 aactcgagat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     4021 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     4081 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttag ttcttgccac
     4141 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     4201 tgacaattcc gtggtgttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4261 ctagctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     4321 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt
     4381 gggaggtttt ttaaagatct ggagaccggt gtggcacgtg cggaccgagc ggccgcagga
     4441 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     4501 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4561 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4621 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4681 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     4741 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4801 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4861 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4921 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4981 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     5041 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     5101 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     5161 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5221 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5281 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5341 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5401 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5461 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5521 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5581 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5641 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5701 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5761 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5821 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5881 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5941 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6001 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6061 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6121 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6181 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6241 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6301 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6361 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6421 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6481 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6541 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6601 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6661 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6721 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6781 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6841 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6901 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6961 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7021 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7081 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7141 ttcctggcct tttgctggcc ttttgctcac atgt
//
