LOCUS       sequence-437814-        7500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1-targeting backbone used as a filler plasmid to co-deliver 
            during nucleofection with pAAV-Puro_siKD2.0 (Plasmid #220536) cloned
            with shRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7500)
  AUTHORS   Balmas E, Ratto ML, Snijders KE, Becca S, Liaci C, Ricca I, Merlo 
            GR, Calogero RA, Alessandri L, Mendjan S, Bertero A
  TITLE     Single Cell Transcriptional Perturbome in Pluripotent Stem Cell 
            Models
  JOURNAL   Mol Syst Biol (2025). https://doi.org/10.1038/s44320-025-00172-8
REFERENCE   2  (bases 1 to 7500)
  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: "Mol Syst 
            Biol (2025). https://doi.org/10.1038/s44320-025-00172-8"
COMMENT     Sequence Label: pAAV-Neo_CAG
FEATURES             Location/Qualifiers
     source          1..7500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    402..1205
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    1212..1237
                     /label=SA
                     /note="splice acceptor site"
     CDS             1261..1314
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1324..2127
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1387..1406)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1997..2016
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2159..2176)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2165..2389
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        2459..2762
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2764..3041
                     /label=chicken beta-actin promoter
     intron          3042..4058
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3982..4004
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4066..4085
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     polyA_signal    4317..4398
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4354..4373)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    4424..5260
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(5279..5295)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5279..5295)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5292..5314)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5303..5319
                     /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)."
     promoter        complement(join(5327..5333,5334..5351,5352..5357))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5372..5393
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5510..5527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5681..6269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5761..5780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6440..7231,7232..7300))
                     /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     7063..7082
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7301..7405)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7473..7491
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtgctttctc tgaccagcat
      421 tctctcccct gggcctgtgc cgctttctgt ctgtagcttg tggcctgggt cacctctacg
      481 gctggcccag atccttccct gccgcctcct tcaggttccg tcttcctcca ctccctcttc
      541 cccttgctct ctgctgtgtt gctgcccaag gatgctcttt ccggagcact tccttctcgg
      601 cgctgcacca cgtgatgtcc tctgagcgga tcctccccgt gtctgggtcc tctccgggca
      661 tctctcctcc ctcacccaac cccatgccgt cttcactcgc tgggttccct tttccttctc
      721 cttctggggc ctgtgccatc tctcgtttct taggatggcc ttctccgacg gatgtctccc
      781 ttgcgtcccg cctccccttc ttgtaggcct gcatcatcac cgtttttctg gacaacccca
      841 aagtaccccg tctccctggc tttagccacc tctccatcct cttgctttct ttgcctggac
      901 accccgttct cctgtggatt cgggtcacct ctcactcctt tcatttgggc agctccccta
      961 ccccccttac ctctctagtc tgtgctagct cttccagccc cctgtcatgg catcttccag
     1021 gggtccgaga gctcagctag tcttcttcct ccaacccggg cccctatgtc cacttcagga
     1081 cagcatgttt gctgcctcca gggatcctgt gtccccgagc tgggaccacc ttatattccc
     1141 agggccggtt aatgtggctc tggttctggg tacttttatc tgtcccctcc accccacagt
     1201 ggggcaagct tctgacctct tctcttcctc ccacagggcc tcgagagatc tggcagcgga
     1261 gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctaggctc
     1321 gagatgggat cggccattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     1381 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     1441 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     1501 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     1561 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     1621 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     1681 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     1741 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     1801 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     1861 cgcatgcccg acggcgatga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     1921 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     1981 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     2041 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     2101 tatcgccttc ttgacgagtt cttctgatct agagggcccg tttaaacccg ctgatcagcc
     2161 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2221 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2281 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2341 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggt cactcgacta
     2401 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     2461 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     2521 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     2581 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     2641 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     2701 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     2761 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
     2821 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg
     2881 gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag
     2941 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
     3001 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
     3061 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
     3121 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
     3181 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
     3241 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
     3301 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
     3361 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
     3421 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
     3481 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
     3541 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
     3601 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
     3661 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
     3721 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
     3781 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
     3841 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     3901 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     3961 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     4021 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     4081 ttgtgctgtc tcatcatttt ggcaaagaat taattcggat caattcgagc tcggtacccg
     4141 gggatcctct agtcagctga cgcgtgctag cgcggccgca tcgataagct tgtcgacgat
     4201 atctctagag gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     4261 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     4321 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4381 aagcattttt ttcactgcct tgacagtact cttaagtcga cttactaggg acaggattgg
     4441 tgacagaaaa gccccatcct taggcctcct ccttcctagt ctcctgatat tgggtctaac
     4501 ccccacctcc tgttaggcag attccttatc tggtgacaca cccccatttc ctggagccat
     4561 ctctctcctt gccagaacct ctaaggtttg cttacgatgg agccagagag gatcctggga
     4621 gggagagctt ggcagggggt gggagggaag ggggggatgc gtgacctgcc cggttctcag
     4681 tggccaccct gcgctaccct ctcccagaac ctgagctgct ctgacgcggc tgtctggtgc
     4741 gtttcactga tcctggtgct gcagcttcct tacacttccc aagaggagaa gcagtttgga
     4801 aaaacaaaat cagaataagt tggtcctgag ttctaacttt ggctcttcac ctttctagtc
     4861 cccaatttat attgttcctc cgtgcgtcag ttttacctgt gagataaggc cagtagccag
     4921 ccccgtcctg gcagggctgt ggtgaggagg ggggtgtccg tgtggaaaac tccctttgtg
     4981 agaatggtgc gtcctaggtg ttcaccaggt cgtggccgcc tctactccct ttctctttct
     5041 ccatccttct ttccttaaag agtccccagt gctatctggg acatattcct ccgcccagag
     5101 cagggtcccg cttccctaag gccctgctct gggcttctgg gtttgagtcc ttggcaagcc
     5161 caggagaggc gctcaggctt ccctgtcccc cttcctcgtc caccatctca tgcccctggc
     5221 tctcctgccc cttccctaca ggggttcctg gctctgctct aagcttggcg taatcatggt
     5281 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5341 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5401 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5521 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5581 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5641 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5701 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5761 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5821 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5881 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5941 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6001 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6061 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6121 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6181 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6241 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6301 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6361 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6421 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6481 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6541 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6601 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6661 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6721 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6781 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6841 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6901 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6961 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7021 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7081 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7141 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7201 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7261 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7321 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7381 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     7441 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
