LOCUS       sequence-384459-        5586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV9 based enhancer activity assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5586)
  AUTHORS   Akerberg BN, Gu F, VanDusen NJ, Zhang X, Dong R, Li K, Zhang B, Zhou
            B, Sethi I, Ma Q, Wasson L, Wen T, Liu J, Dong K, Conlon FL, Zhou J,
            Yuan GC, Zhou P, Pu WT
  TITLE     A reference map of murine cardiac transcription factor chromatin 
            occupancy identifies dynamic and conserved enhancers.
  JOURNAL   Nat Commun. 2019 Oct 28;10(1):4907. doi: 10.1038/s41467-019-12812-3.
  PUBMED    31659164
REFERENCE   2  (bases 1 to 5586)
  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: "Nat 
            Commun."; date: "2019-10-28"; volume: "10(1):4907. doi"; pages: " 
            10.1038/s41467-019-12812-3"
COMMENT     Sequence Label: aav-U6-broccoli-hsp68-mCherry-MCS-pA
FEATURES             Location/Qualifiers
     source          1..5586
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        198..446
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     198..218
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     369..388
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             1620..2330
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1765..1783)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2033..2052
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2421..2440)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2486..2541
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   2634..2763
                     /label=AAV2 ITR
     primer_bind     complement(2782..2799)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2782..2798)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2791..2813)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2940..3395
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3027..3046)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3237..3258
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3421..3525
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3526..3594,3595..4386)
                     /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(3744..3763)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4557..5145
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5046..5065
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5433..5454
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5469..5474,5475..5492,5493..5499)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5507..5523
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5512..5534
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5531..5547
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5531..5547
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcggcctc agtgagcgag
       61 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttccttgta gttaatgatt
      121 aacccgccat gctacttatc taccagggta atggggatcc tctagaacta tagctagaat
      181 tcaaggtcgg gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat
      241 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
      301 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
      361 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
      421 atatcttgtg gaaaggacga aacaccgtgc tcgcttcggc agcacatata ctagtcgact
      481 tgccatgtgt atgtgggaga cggtcgggtc catctgagac ggtcgggtcc agatattcgt
      541 atctgtcgag tagagtgtgg gctcagatgt cgagtagagt gtgggctccc acatactctg
      601 atgatccaga cggtcgggtc catctgagac ggtcgggtcc agatattcgt atctgtcgag
      661 tagagtgtgg gctcagatgt cgagtagagt gtgggctgga tcattcatgg caatctagag
      721 cggacttcgg tccgcttttt ggtaccgagc tccaggaaca tccaaactga gcagccgggg
      781 tcccccccac cccccacccc gcccctcccg gcaactttga gcctgtgctg ggacagagcc
      841 tctagttcct aaattagtcc atgaggtcag aggcagcact gccattgtaa cgcgattgga
      901 gaggatcacg tcaccggaca cgcccccagg catctccctg ggtctcctaa acttggcggg
      961 gagaagtttt agcccttaag ttttagcctt taacccccat attcagaact gtgcgagttg
     1021 gcgaaacccc acaaatcaca acaaactgta cacaacaccg agctagaggt gatctttctt
     1081 gtccattcca cacaggcctt agtaatgcgt cgccatagca acagtgtcac tagtagcacc
     1141 agcacttccc cacaccctcc ccctcaggaa tccgtactct ccagtgaacc ccagaaacct
     1201 ctggagagtt ctggacaagg gcggaaccca caactccgat tactcaaggg aggcggggaa
     1261 gctccaccag acgcgaaact gctggaagat tcctggcccc aaggcctcct ccggctcgct
     1321 gattggccca gcggagagtg ggcggggccg gtgaagactc cttaaaggcg caggacggcg
     1381 agcaggtcac cagacgctga cagctactca gaaccaaatc tggttccatc cagagacaag
     1441 cgaagacaag agaagcagag cgagcggcgc gttcccgatc ctcggccagg accagccttc
     1501 cccagagcat ccctgccgcg gagcgcaacc ttcccaggag catccctgcc gcggagcgca
     1561 actttccccg gagcatccac gccgcggagc gcagccttcc agaagcagag cgcggcgcca
     1621 tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     1681 tgcacatgga gggctccgtg aacggccacg agtttgagat tgaaggcgaa ggcgagggca
     1741 gaccctacga gggaacacag accgccaagc tgaaggtgac caagggtggc cccctgccct
     1801 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
     1861 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     1921 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     1981 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     2041 tgcagaagaa gacaatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     2101 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
     2161 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
     2221 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     2281 gcgccgaggg tcgacactcc accggcggca tggacgagct gtacaagtag ctgcggccgc
     2341 atcagagcta gcttcagcat cgatacttga ggcgcgccta gccatctcga ggacggggtg
     2401 aactacgcct gaggatccga tctttttccc tctgccaaaa attatgggga catcatgaag
     2461 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     2521 tggaattttt tgtgtctctc actcggaagc aattcgttga tctgaatttc gaccacccat
     2581 aatacccatt accctggtag ataagtagca tggcgggtta atcattaact acaaggaacc
     2641 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     2701 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     2761 cagccttaat taacctaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2821 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     2881 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     2941 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3001 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3061 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3121 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3181 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3241 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3301 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3361 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     3421 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3481 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3541 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3601 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3661 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3721 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3781 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3841 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3901 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3961 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4021 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     4081 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     4141 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4201 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     4261 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4321 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4381 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4441 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4501 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4561 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4621 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4681 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4741 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4801 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4861 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4921 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4981 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5041 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5101 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5161 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5221 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     5281 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     5341 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     5401 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     5461 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     5521 caatttcaca caggaaacag ctatgaccat gattacgcca gatttaatta aggccttaat
     5581 taggct
//
