LOCUS       Exported                6986 bp ds-DNA     circular SYN 09-JUN-2025
DEFINITION  Empty vector for astrocyte-selective expression in AAV; astrocyte 
            specificity enhanced with 4x6T miRNA targeting cassette.
ACCESSION   .
VERSION     .
KEYWORDS    AAV-GfaABC1D-DIO-Lck-mcherry-ibARK(D110A)-4x6T-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6986)
  TITLE     A toolbox of astrocyte-specific, serotype-independent 
            adeno-associated viral vectors using microRNA targeting sequences
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6986)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jun 11, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6986
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1506..3021
     rep_origin      23..611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     512..531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   673..802
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     complement(695..706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(696..711)
                     /label=No.37_Addgene-ITR_AAV2_Rv
     primer_bind     740..755
                     /label=No.37_Addgene-ITR_AAV2_Rv
     primer_bind     745..756
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(781..801)
                     /label=No.36_Addgene-ITR_AAV2_Fw
     primer_bind     782..802
                     /label=No.35_ITR-primer-Fw&Rv
     primer_bind     complement(1161..1172)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     1414..1434
                     /label=No56. GfaABC1D promotor
     primer_bind     1491..1522
                     /label=In-fusion to JN112 Fw.
     misc_feature    1523..1556
                     /label=Lox2272
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Recognition site of Cre 
                     recombinase;
                     can't work with other types of Lox site."
                     /note="/description=Mutated Lox site with two base 
                     substitutions
                     of LoxP"
                     /note="/official_designation=Lox2272"
                     /note="/uuid=a19dd1e203df40eb846e5102df13ffb4"
     misc_feature    1589..1622
                     /label=LoxP
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Recognition site of Cre 
                     recombinase."
                     /note="/description=Locus of X(cross)-over in P1"
                     /note="/official_designation=LoxP"
                     /note="/uuid=b48e25a55648467f8c98b3f68faf613f"
     CDS             complement(1635..2891)
                     /codon_start=1
                     /label={Lck-mcherry-ibARK(D110A)}
                     /label={Lck-mcherry-ibARK}
                     /note="/application_notes="
                     /note="/official_designation={Lck-mcherry-ibARK}"
                     /note="/uuid=d9156fb42d874565836c73490f4bdad3"
                     /translation="MGCGCSSNPEDDWMENIDVCENCHYPPPKLRIDPPVATMVSKGEE
                     DNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDIL
                     SPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIY
                     KVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKT
                     TYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKKYLEDRG
                     EVTFEKIFSQKLGYLLFRDFCLKHLEEAKPLVEFYEEIKKYEKLETEEERLVCSREIFA
                     TYIMKELLACSHPFSKSAIEHVQGHLVKKQVPPDLFQPYIEEICQNLRGDVFQKFIESD
                     KFTRFCQWKNVELNIHGSG"
     primer_bind     2070..2084
                     /label=No.73 FLEX-iBARK top
     primer_bind     complement(2345..2364)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2345..2364)
                     /label=No.140 mCherry-F
     primer_bind     2614..2632
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    complement(2892..2897)
                     /label=Kozak
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Facilitates translation 
                     initiation of
                     ATG start codon downstream of the Kozak sequence."
                     /note="/description=Kozak translation initiation sequence"
                     /note="/full_name=Kozak"
                     /note="/official_designation=Kozak"
                     /note="/uuid=ba4f837cfff042e38e7561244b24cfa6"
     misc_feature    complement(2904..2937)
                     /label=Lox2272
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Recognition site of Cre 
                     recombinase;
                     can't work with other types of Lox site."
                     /note="/description=Mutated Lox site with two base 
                     substitutions
                     of LoxP"
                     /note="/official_designation=Lox2272"
                     /note="/uuid=991e0551b2034abd9c3ad4e364260ba6"
     misc_feature    complement(2970..3003)
                     /label=LoxP
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Recognition site of Cre 
                     recombinase."
                     /note="/description=Locus of X(cross)-over in P1"
                     /note="/official_designation=LoxP"
                     /note="/uuid=f4dc79ba31354e87bba6736ab88dbb23"
     primer_bind     complement(3004..3036)
                     /label=In-fusion to JN112 Rv.
     primer_bind     complement(3624..3642)
                     /label=No.50 mScarlet-I-Rv
     misc_feature    3750..4338
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     3750..3765
                     /label=No.41_WPRE-Fw-1
     primer_bind     complement(3760..3780)
                     /label=No24.WPRE-sec check
     primer_bind     complement(3803..3823)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(3956..3973)
                     /label=プライマー 1
     CDS             complement(4221..4232)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4396..4872
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4595..4614)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4921..5061
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4921..5050
                     /label=AAV2 ITR
     primer_bind     complement(4921..4941)
                     /label=No.35_ITR-primer-Fw&Rv
     primer_bind     4922..4942
                     /label=No.36_Addgene-ITR_AAV2_Fw
     primer_bind     complement(4967..4978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4968..4983)
                     /label=No.37_Addgene-ITR_AAV2_Rv
     primer_bind     5023..5038
                     /label=No.37_Addgene-ITR_AAV2_Rv
     primer_bind     5028..5039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5136..5591
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5223..5242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5433..5454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5608..5627)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5727..5749
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5787..5805)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5873..5977
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5978..6838
                     /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(6196..6215)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
       61 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
      361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      661 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
      721 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      781 aactccatca ctaggggttc ctgcggccgc acgcgtacat atcctggtgt ggagtagggg
      841 acgctgctct gacagaggct cgggggcctg agctggctct gtgagctggg gaggaggcag
      901 acagccaggc cttgtctgca agcagacctg gcagcattgg gctggccgcc ccccagggcc
      961 tcctcttcat gcccagtgaa tgactcacct tggcacagac acaatgttcg gggtgggcac
     1021 agtgcctgct tcccgccgca ccccagcccc cctcaaatgc cttccgagaa gcccattgag
     1081 cagggggctt gcattgcacc ccagcctgac agcctggcat cttgggataa aagcagcaca
     1141 gccccctagg ggctgccctt gctgtgtggc gccaccggcg gtggagaaca aggctctatt
     1201 cagcctgtgc ccaggaaagg ggatcagggg atgcccaggc atggacagtg ggtggcaggg
     1261 ggggagagga gggctgtctg cttcccagaa gtccaaggac acaaatgggt gaggggagag
     1321 ctctccccat agctgggctg cggcccaacc ccaccccctc aggctatgcc agggggtgtt
     1381 gccaggggca cccgggcatc gccagtctag cccactcctt cataaagccc tcgcatccca
     1441 ggagcgagca gagccagagc aggttggaga ggagacgcat cacctccgct gctcgcttaa
     1501 ttaaggtaca aaaaagcagg ctataacttc gtataggata ctttatacga agttatcctc
     1561 gagtaccggt cacgcgtacg caagcttgat aacttcgtat agcatacatt atacgaagtt
     1621 atcctagggc caccctagcc actaccgtgg atgttgagct ctacattctt ccactggcaa
     1681 aatcgtgtga atttatcgct ctcgatgaat ttctggaaca cgtctcctcg gaggttctgg
     1741 caaatttctt caatatatgg ctggaagaga tccggaggca cctgcttctt caccagatgg
     1801 ccctggacgt gctcaatggc gctcttcgag aaaggatgtg agcaggccag cagctccttc
     1861 atgatgtacg tggcgaagat ctctcggctg cagaccaggc gctcctcctc tgtctccagc
     1921 ttctcgtatt tcttgatctc ctcgtagaac tctaccaagg gcttggcctc ctccaggtgc
     1981 ttcaggcaga agtctcggaa aagcaggtac cccagcttct gggagaagat cttctcaaaa
     2041 gtcacctcgc cccggtcctc caggtacttc ttgtacagct cgtccatgcc gccggtggag
     2101 tggcggccct cggcgcgttc gtactgttcc acgatggtgt agtcctcgtt gtgggaggtg
     2161 atgtccaact tgatgttgac gttgtaggcg ccgggcagct gcacgggctt cttggccttg
     2221 taggtggtct tgacctcagc gtcgtagtgg ccgccgtcct tcagcttcag cctctgcttg
     2281 atctcgccct tcagggcgcc gtcctcgggg tacatccgct cggaggaggc ctcccagccc
     2341 atggtcttct tctgcattac ggggccgtcg gaggggaagt tggtgccgcg cagcttcacc
     2401 ttgtagatga actcgccgtc ctgcagggag gagtcctggg tcacggtcac cacgccgccg
     2461 tcctcgaagt tcatcacgcg ctcccacttg aagccctcgg ggaaggacag cttcaagtag
     2521 tcggggatgt cggcggggtg cttcacgtag gccttggagc cgtacatgaa ctgaggggac
     2581 aggatgtccc aggcgaaggg cagggggcca cccttggtca ccttcagctt ggcggtctgg
     2641 gtgccctcgt aggggcggcc ctcgccctcg ccctcgatct cgaactcgtg gccgttcacg
     2701 gagccctcca tgtgcacctt gaagcgcatg aactccttga tgatggccat gttatcctcc
     2761 tcgcccttgc tcaccatggt ggcgaccggt ggatcaattc gaagcttcgg cgggggataa
     2821 tggcagttct cacacacgtc aatgttctcc atccagtcat cttcagggtt tgagctgcag
     2881 ccacagccca tggtggctct agaataactt cgtataaagt atcctatacg aagttatagg
     2941 tacctgagct caacatcgat agcactagta taacttcgta taatgtatgc tatacgaagt
     3001 tatacccagc tttcttgtac acgctagcca attgggatcc atcgattgat caactagtca
     3061 tatgggcatt caccgcgtgc cttactacgc gtattcttaa gcaataagcg aatataacac
     3121 ggtcgatcta aaaaggttag ctgggtgtgt tagaagccct gcaagacgac ctgcgcatta
     3181 ttactcacgg tacgacgcat tattactcac ggtacgaggc attcaccgcg tgccttacta
     3241 cgcgtattct taagcaataa gcgaatataa cacggtcgat ctaaaaaggt tagctgggtg
     3301 tgttagaagc cctgcaagac gacctgggca ttcaccgcgt gccttactac gcgtattctt
     3361 aagcaataag cgaatataac acggtcgatc taaaaaggtt agctgggtgt gttagaagcc
     3421 ctgcaagacg acctgggtac cgcattatta ctcacggtac gacgcattat tactcacggt
     3481 acgaggcatt caccgcgtgc cttactacgc gtattcttaa gcaataagcg aatataacac
     3541 ggtcgatcta aaaaggttag ctgggtgtgt tagaagccct gcaagacgac ctgggtacca
     3601 ggtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     3661 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattct
     3721 gcctcgagca gcgatatcat accgtcgaca atcaacctct ggattacaaa atttgtgaaa
     3781 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3841 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3901 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3961 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4021 tttccgggac tttcgctttc cccctcccta ttgccacggt ggaactcatc gccgcctgtc
     4081 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     4141 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     4201 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     4261 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     4321 tttgggccgc ctccccgcct gtctagagtc gacctgcaga agcttgcctc gagcagcgct
     4381 gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg
     4441 gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg
     4501 tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg
     4561 caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca
     4621 gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct
     4681 cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt
     4741 ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag
     4801 gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc
     4861 ttccctgtcc ttctgatttt gtaggtaacc acgtggtggt ggtgcggacc gagcggccgc
     4921 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4981 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     5041 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     5101 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     5161 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     5221 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5281 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5341 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5401 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5461 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     5521 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5581 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5641 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5701 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5761 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5821 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     5881 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5941 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6001 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6061 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6121 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6181 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6241 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6301 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6361 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6421 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6481 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6541 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6601 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6661 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6721 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6781 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6841 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6901 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6961 ttcgttccac tgagcgtcag accccg
//
