LOCUS       sequence-491420-        6704 bp    DNA     circular SYN 03-NOV-2025
DEFINITION  3' AAVLINK plasmid for KDM6B expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6704)
  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  JOURNAL   Unpublished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 6704)
  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            "Unpublished"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'-KDM6B-FLAG
FEATURES             Location/Qualifiers
     source          1..6704
                     /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             3412..3477
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3554..3574)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3967..4107
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3967..4096
                     /label=AAV2 ITR
     rep_origin      4182..4637
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4269..4288)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4479..4500
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4654..4673)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4773..4795
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4833..4851)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4919..5023
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5024..5092,5093..5884)
                     /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(5242..5261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6055..6643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6544..6563
                     /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 gggcaaaggc caaggccaag
     1501 gtccccaaag aaaagagccg ccgggtgctg gggaacctgg acctgcagag cgaggagatc
     1561 cagggtcgtg agaagtcccg gcccgatctt ggcggggcct ccaaggccaa gccacccaca
     1621 gctccagccc ctccatcagc tcctgcacct tctgcccagc ccacaccccc gtcagcctct
     1681 gtccctggaa agaaggctcg ggaggaagcc ccagggccac cgggtgtcag ccgggccgac
     1741 atgctgaagc tgcgctcact tagtgagggg ccccccaagg agctgaagat ccggctcatc
     1801 aaggtagaga gtggtgacaa ggagaccttt atcgcctctg aggtggaaga gcggcggctg
     1861 cgcatggcag acctcaccat cagccactgt gctgctgacg tcgtgcgcgc cagcaggaat
     1921 gccaaggtga aagggaagtt tcgagagtcc tacctttccc ctgcccagtc tgtgaaaccg
     1981 aagatcaaca ctgaggagaa gctgccccgg gaaaaactca acccccctac acccagcatc
     2041 tatctggaga gcaaacggga tgccttctca cctgtcctgc tgcagttctg tacagaccct
     2101 cgaaatccca tcacagtgat ccggggcctg gcgggctccc tgcggctcaa cttgggcctc
     2161 ttctccacca agaccctggt ggaagcgagt ggcgaacaca ccgtggaagt tcgcacccag
     2221 gtgcagcagc cctcagatga gaactgggat ctgacaggca ctcggcagat ctggccttgt
     2281 gagagctccc gttcccacac caccattgcc aagtacgcac agtaccaggc ctcatccttc
     2341 caggagtctc tgcaggagga gaaggagagt gaggatgagg agtcagagga gccagacagc
     2401 accactggaa cccctcctag cagcgcacca gacccgaaga accatcacat catcaagttt
     2461 ggcaccaaca tcgacttgtc tgatgctaag cggtggaagc cccagctgca ggagctgcta
     2521 aagctgcccg ccttcatgcg ggtaacatcc acgggcaaca tgctgagcca cgtgggccac
     2581 accatcctgg gcatgaacac ggtgcagctg tacatgaagg tgcccggcag ccgaacgcca
     2641 ggccaccagg agaataacaa cttctgctcc gtcaacatca acattggccc aggcgactgc
     2701 gagtggttcg cggtgcacga gcactactgg gagaccatca gcgctttctg tgatcggcac
     2761 ggcgtggact acttgacggg ttcctggtgg ccaatcctgg atgatctcta tgcatccaat
     2821 attcctgtgt accgcttcgt gcagcgaccc ggagacctcg tgtggattaa tgcggggact
     2881 gtgcactggg tgcaggccac cggctggtgc aacaacattg cctggaacgt ggggcccctc
     2941 accgcctatc agtaccagct ggccctggaa cgatacgagt ggaatgaggt gaagaacgtc
     3001 aaatccatcg tgcccatgat tcacgtgtca tggaacgtgg ctcgcacggt caaaatcagc
     3061 gaccccgact tgttcaagat gatcaagttc tgcctgctgc agtccatgaa gcactgccag
     3121 gtgcaacgcg agagcctggt gcgggcaggg aagaaaatcg cttaccaggg ccgtgtcaag
     3181 gacgagccag cctactactg caacgagtgc gatgtggagg tgtttaacat cctgttcgtg
     3241 acaagtgaga atggcagccg caacacgtac ctggtacact gcgagggctg tgcccggcgc
     3301 cgcagcgcag gcctgcaggg cgtggtggtg ctggagcagt accgcactga ggagctggct
     3361 caggcctacg acgccttcac gctggcccca gccagcacgt cgcgatgtac agactacaaa
     3421 gaccatgacg gtgattataa agatcatgac atcgactaca aggatgacga tgacaagtaa
     3481 gaattctgat gactcgagat aatcaacctc tggattacaa aatttgtgaa agattgactg
     3541 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     3601 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttag
     3661 ttcttgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3721 tgttgggcac tgacaattcc gtggtgttta tttgtgaaat ttgtgatgct attgctttat
     3781 ttgtaaccat ctagctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     3841 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     3901 ggggagatgt gggaggtttt ttaaagatct ggagaccggt gtggcacgtg cggaccgagc
     3961 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4021 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4081 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4141 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4201 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     4261 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4321 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4381 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4441 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4501 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     4561 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4621 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4681 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4741 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4801 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     4861 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     4921 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4981 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5041 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5101 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5161 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5221 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5281 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5341 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5401 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5461 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5521 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5581 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5641 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5701 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5761 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5821 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5881 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5941 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6001 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6061 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6121 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6181 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6241 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6301 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6361 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6421 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6481 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6541 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6601 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6661 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
