LOCUS       sequence-361415-        6755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To produce AAV with the CBh promoter to express IgkL-mNeonGreen 
            (IgKL is a secretory signal peptide).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6755)
  AUTHORS   Wang X, Delle C, Asiminas A, Akther S, Vittani M, Brogger P, Kusk P,
            Vo CT, Radovanovic T, Konno A, Hirai H, Fukuda M, Weikop P, Goldman 
            SA, Nedergaard M, Hirase H
  TITLE     Liver-secreted fluorescent blood plasma markers enable chronic 
            imaging of the microcirculation.
  JOURNAL   Cell Rep Methods. 2022 Sep 21;2(10):100302. doi: 
            10.1016/j.crmeth.2022.100302. eCollection 2022 Oct 24.
  PUBMED    36313804
REFERENCE   2  (bases 1 to 6755)
  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: "Cell Rep 
            Methods."; date: "2022-09-21"; volume: "2(10):100302. doi"; pages: "
            10.1016/j.crmeth.2022.100302. eCollection 2022 Oct 24"
COMMENT     Sequence Label: pAAV-CBh-IgKL-mNG
FEATURES             Location/Qualifiers
     source          1..6755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        124..409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        411..685
                     /label=chicken beta-actin promoter
     intron          686..913
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             991..1698
                     /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      1243..1252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1719..2307
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1772..1792)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2190..2201)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2434..2828
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     repeat_region   3112..3241
                     /label=AAV2 ITR
     rep_origin      3612..4125
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3699..3718)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3909..3930
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4607..4626)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4726..4748
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4786..4804)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4872..4976
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4977..5045,5046..5837)
                     /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(5195..5214)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6008..6596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6497..6516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg
       61 tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag actcgagacg
      121 cgtcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      181 attgacgtca atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      241 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      301 tgacgtcaat gacggtaaat ggcccgcctg gcattgtgcc cagtacatga ccttatggga
      361 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag
      421 ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt
      481 atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggc gcgcgccagg
      541 cggggcgggg cggggsgrgg ggsggggsgg ggsgaggcgg rgaggtgcgg cggcagccaa
      601 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      661 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
      721 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
      781 gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt
      841 taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat
      901 cacttttttt caggttggac cggtgccacc atgacagaca cactcctgct atgggtactg
      961 ctgctctggg ttccaggttc cactggtgac gtgagcaagg gcgaggagga taacatggcc
     1021 tctctcccag cgacacatga gttacacatc tttggctcca tcaacggtgt ggactttgac
     1081 atggtgggtc agggcaccgg caatccaaat gatggttatg aggagttaaa cctgaagtcc
     1141 accaagggtg acctccagtt ctccccctgg attctggtcc ctcatatcgg gtatggcttc
     1201 catcagtacc tgccctaccc tgacgggatg tcgcctttcc aggccgccat ggtagatggc
     1261 tccggctacc aagtccatcg cacaatgcag tttgaagatg gtgcctccct tactgttaac
     1321 taccgctaca cctacgaggg aagccacatc aaaggagagg cccaggtgaa ggggactggt
     1381 ttccctgctg acggtcctgt gatgaccaac tcgctgaccg ctgcggactg gtgcaggtcg
     1441 aagaagactt accccaacga caaaaccatc atcagtacct ttaagtggag ttacaccact
     1501 ggaaatggca agcgctaccg gagcactgcg cggaccacct acacctttgc caagccaatg
     1561 gcggctaact atctgaagaa ccagccgatg tacgtgttcc gtaagacgga gctcaagcac
     1621 tccaagaccg agctcaactt caaggagtgg caaaaggcct ttaccgatgt gatgggcatg
     1681 gacgagctgt acaagtaaag ctaagcggcc gcatcgataa tcaacctctg gattacaaaa
     1741 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     1801 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     1861 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     1921 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     1981 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     2041 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     2101 tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc
     2161 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     2221 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     2281 ggatctccct ttgggccgcc tccccgcatc gatgccgtat acggtacccg gggatcctct
     2341 agtagagtcg aggaattcac cccaccagtg caggctgcct atcagaaagt ggtggctggt
     2401 gtggctaatg ccctggccca caagtatcac taagctcgct ttcttgctgt ccaatttcta
     2461 ttaaaggttc ctttgttccc taagtccaac tactaaactg ggggatatta tgaagggcct
     2521 tgagcatctg gattctgcct aataaaaaac atttattttc attgcaatga tgtatttaaa
     2581 ttatttctga atattttact aaaaagggaa tgtgggaggt cagtgcattt aaaacataaa
     2641 gaaatgaaga gctagttcaa accttgggaa aatacactat atcttaaact ccatgaaaga
     2701 aggtgaggct gcaaacagct aatgcacatt ggcaacagcc cctgatgcct atgccttatt
     2761 catccctcag aaaaggattc aagtagaggc ttgatttgga ggttaaagtt ttgctatgct
     2821 gtattttaca ttacttattg ttttagctgt cctcatgaat gtcttttcac tacccatttg
     2881 cttatcctgc atctctcagc cttgactcca ctcagttctc ttgcttagag ataccacctt
     2941 tcccctgaag tgttccttcc atgttttacg gcgagatggt ttctcctcgc ctggccactc
     3001 agccttagtt gtctctgttg tcttatagag gtctggtcga cccccaacgc gttggagctc
     3061 tagagcatgg ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc
     3121 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     3181 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     3241 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     3301 atggcgaatg gcgattccgt tgcaatggct ggcggtaata ttgttctgga tattaccagc
     3361 aaggccgata gtttgagttc ttctactcag gcaagtgatg ttattactaa tcaaagaagt
     3421 attgcgacaa cggttaattt gcgtgatgga cagactcttt tactcggtgg cctcactgat
     3481 tataaaaaca cttctcagga ttctggcgta ccgttcctgt ctaaaatccc tttaatcggc
     3541 ctcctgttta gctcccgctc tgattctaac gaggaaagca cgttatacgt gctcgtcaaa
     3601 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3661 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3721 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3781 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3841 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3901 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3961 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4021 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttaaa tatttgctta
     4081 tacaatcttc ctgtttttgg ggcttttctg attatcaacc ggggtacata tgattgacat
     4141 gctagtttta cgattaccgt tcatcgattc tcttgtttgc tccagactct caggcaatga
     4201 cctgatagcc tttgtagaga cctctcaaaa atagctaccc tctccggcat gaatttatca
     4261 gctagaacgg ttgaatatca tattgatggt gatttgactg tctccggcct ttctcacccg
     4321 tttgaatctt tacctacaca ttactcaggc attgcattta aaatatatga gggttctaaa
     4381 aatttttatc cttgcgttga aataaaggct tctcccgcaa aagtattaca gggtcataat
     4441 gtttttggta caaccgattt agctttatgc tctgaggctt tattgcttaa ttttgctaat
     4501 tctttgcctt gcctgtatga tttattggat gttggaatcg cctgatgcgg tattttctcc
     4561 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     4621 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4681 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4741 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     4801 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4861 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4921 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4981 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5041 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5101 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5161 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5221 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5281 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5341 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5401 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5461 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5521 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5581 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5641 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5701 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5761 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5821 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5881 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5941 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6001 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6061 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6121 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6181 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6241 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6301 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6361 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6421 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6481 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6541 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6601 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6661 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6721 ccgctcgccg cagccgaacg accgagcgca gcgag
//
