LOCUS       sequence-397551-        6102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knocks in mNeonGreen into exon 14 of Alb to produce Alb-mNeonGreen 
            fusion protein in mice.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6102)
  AUTHORS   Vittani M, Knak PAG, Fukuda M, Nagao M, Wang X, Kjaerby C, Konno A, 
            Hirai H, Nedergaard M, Hirase H
  TITLE     Virally induced CRISPR/Cas9-based knock-in of fluorescent albumin 
            allows long-term visualization of cerebral circulation in infant and
            adult mice.
  JOURNAL   Fluorescence Imaging of the Brain. Neuromethods, vol 209. Humana, 
            New York, NY.
REFERENCE   2  (bases 1 to 6102)
  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: 
            "Fluorescence Imaging of the Brain. Neuromethods, vol 209. Humana, 
            New York, NY."
COMMENT     Sequence Label: pAAV-U6-AlbEx14-mNG
FEATURES             Location/Qualifiers
     source          1..6102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        139..387
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     139..159
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     310..329
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        409..484
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(496..520)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1583..2290
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      1835..1844
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     repeat_region   3296..3436
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3296..3425
                     /label=AAV2 ITR
     rep_origin      3511..3966
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3598..3617)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3808..3829
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3983..4002)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4102..4124
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4162..4180)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4248..4352
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4353..4421,4422..5213)
                     /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(4571..4590)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5384..5972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5873..5892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6034..6102,1..61)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc
       61 tgcggccgct gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac tggatccggt
      121 accaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga
      181 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
      241 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
      301 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
      361 tatatcttgt ggaaaggacg aaacaccgct tctcaggtaa ctatacttgt tttagagcta
      421 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      481 gtgctttttt tctagaccca gctttcttgt acaaagttgg cattactgaa actagacaaa
      541 acccgtgtga ctggcatcga ttattctatt tgatctagct agtcctagca aagtgacaac
      601 tgctactccc ctcctacaca gccaagattc ctaagttggc agtggcatgc ttaatcctca
      661 aagccaaagt tacttggctc caagatttat agccttaaac tgtggcctca cattccttcc
      721 tatcttactt tcctgcactg gggtaaatgt ctccttgctc ttcttgcttt ctgtcctact
      781 gcagggctct tgctgagctg gtgaagcaca agcccaaggc tacagcggag caactgaaga
      841 ctgtcatgga tgactttgca cagttcctgg atacatgttg caaggctgct gacaaggaca
      901 cctgcttctc gactgaggtc agaaacgttt ttgcattttg acgatgttca gtttccattt
      961 tctgtgcacg tggtcaggtg tagctctctg gaactcacac actgaataac tccaccaatc
     1021 tagatgttgt tctctacgta actgtaatag aaactgactt acgtagcttt taatttttat
     1081 tttctgccac actgctgcct attaaatacc tattatcact atttggtttc aaatttgtga
     1141 cacagaagag catagttaga aatacttgca aagcctagaa tcatgaactc atttaaacct
     1201 tgccctgaaa tgtttctttt tgaattgagt tattttacac atgaatggac agttaccatt
     1261 atatatctga atcatttcac attccctccc atggcctaac aacagtttat cttcttattt
     1321 tgggcacaac agatgtcaga gagcctgctt taggaattct aagtagaact gtaattaagc
     1381 aatgcaaggc acgtacgttt actatgtcat tgcctatggc tatgaagtgc aaatcctaac
     1441 agtcctgcta atacttttct aacatccatc atttctttgt tttcagggtc caaagtttgt
     1501 cactagatgc aaagacgcct tagcccccgg gggtggaggc ggttcaggcg gaggtggctc
     1561 tggcggtggc ggatcagtcg acgtgagcaa gggcgaggag gataacatgg cctctctccc
     1621 agcgacacat gagttacaca tctttggctc catcaacggt gtggactttg acatggtggg
     1681 tcagggcacc ggcaatccaa atgatggtta tgaggagtta aacctgaagt ccaccaaggg
     1741 tgacctccag ttctccccct ggattctggt ccctcatatc gggtatggct tccatcagta
     1801 cctgccctac cctgacggga tgtcgccttt ccaggccgcc atggtagatg gctccggata
     1861 ccaagtccat cgcacaatgc agtttgaaga tggtgcctcc cttactgtta actaccgcta
     1921 cacctacgag ggaagccaca tcaaaggaga ggcccaggtg aaggggactg gtttccctgc
     1981 tgacggtcct gtgatgacca actcgctgac cgctgcggac tggtgcaggt cgaagaagac
     2041 ttaccccaac gacaaaacca tcatcagtac ctttaagtgg agttacacca ctggaaatgg
     2101 caagcgctac cggagcactg cgcggaccac ctacaccttt gccaagccaa tggcggctaa
     2161 ctatctgaag aaccagccga tgtacgtgtt ccgtaagacg gagctcaagc actccaagac
     2221 cgagctcaac ttcaaggagt ggcaaaaggc ctttaccgat gtgatgggca tggacgagct
     2281 gtacaagtaa acacatcaca accacaacct tctcaggtaa ctatacttga cacttaaaaa
     2341 acataatcat aatcattttt cctaaaacga tcaagactga taaccatttg acaagagcca
     2401 tacagacaag caccagctgg cactcttagg tcttcacgta tggtcatcag tttgggttcc
     2461 atttgtagat aagaaactga acatataaag gtctaggtta atgcaattta cacaaaagga
     2521 gaccaaacca gggagagaag gaaccaaaat taaaaattca aaccagagca aaggagttag
     2581 ccctggtttt gctctgactt acatgaacca ctatgtggag tcctccatgt tagcctagtc
     2641 aagcttatcc tctggatgaa gttgaaacca tatgaaggaa tatttggggg gtgggtcaaa
     2701 acagttgtgt atcaatgatt ccatgtggtt tgacccaatc attctgtgaa tccatttcaa
     2761 cagaagatac aacgggttct gtttcataat aagtgatcca cttccaaatt tctgatgtgc
     2821 cccatgctaa gctttaacag aatttatctt cttatgacaa agcagcctcc tttgaaaata
     2881 tagccaactg cacacagcta tgttgatcaa ttttgtttat aatcttgcag aagagaattt
     2941 tttaaaatag ggcaataatg gaaggctttg gcaaaaaaat tgtttctcca tatgaaaaca
     3001 aaaaacttat ttttttattc aagcaaagaa cctatagaca taaggctatt tcaaaattat
     3061 ttcagtttta gaaagaattg aaagttttgt agcattctga gaagacagct ttcatttgta
     3121 atcataggta atatgtaggt cctcagaaat ggtgagaccc ctgactttga cacttgggga
     3181 ctctgaggga ccagtgatga agagggcaca acttatatca cacatgcacg agttggggtg
     3241 agagggtgtc acaacatcta tcagtgtgtc atctgcccac caagtaagcg gccgcaggaa
     3301 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     3361 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     3421 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3481 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     3541 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     3601 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3661 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3721 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3781 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     3841 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     3901 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     3961 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     4021 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4081 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4141 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     4201 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     4261 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     4321 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     4381 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     4441 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     4501 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     4561 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     4621 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     4681 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     4741 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     4801 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     4861 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     4921 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     4981 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5041 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5101 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     5161 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     5221 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     5281 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5341 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5401 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5461 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5521 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5581 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5641 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5701 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5761 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5821 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5881 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5941 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6001 tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc
     6061 tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc cg
//
