LOCUS       pAAV2-sA-NeoR           6237 bp    DNA     circular SYN 23-MAY-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6237)
  AUTHORS   Hart Lab
  TITLE     Direct Submission
  JOURNAL   Exported May 23, 2025 from SnapGene 8.0.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          join(2238..6237,1..2237)
                     /plasmid="pAAVS1-DIO-rev-dCas9-KRAB-P2A-EGFP"
                     /mol_type="other DNA"
                     /note="color: #ffffff"
                     /organism="synthetic DNA construct"
     misc_feature    1..804
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
                     /note="color: #ffe4c4"
     misc_feature    811..836
                     /label=SA
                     /note="splice acceptor site"
                     /note="color: #a6acb3
                     Cleavage site after base 835"
     CDS             860..913
                     /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."
                     /note="color: #cc99b2
                     Cleavage site after base 910"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             929..1732
                     /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))"
                     /note="color: #ccffcc"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2237)
                     /label=pAAVS1 vector_rev
                     /note="pAAVS1 vector_rev"
                     /note="gene-specific Tm: 58.7  Ta: 59.7"
                     /note="gene-specific primer: 
                     CCGTAATTGATTACTATTAATAACTAGTTC"
                     /note="color: #a020f0; direction: LEFT"
     gene            join(2238..6237,1..2237)
                     /label=pAAVS1 vector
                     /note="pAAVS1 vector"
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    2248..3084
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
                     /note="color: #ffe4c4"
     promoter        complement(3119..3137)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(3144..3160)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     CDS             3298..3600
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /note="color: #993366"
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             complement(join(4190..4981,4982..5050))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 4190 .. 4981 / #ccffcc
                        2: 4982 .. 5050 / #ccffcc / signal sequence
                     Cleavage site after base 4981"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5174..5762
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     protein_bind    6050..6071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     promoter        join(6086..6091,6092..6109,6110..6116)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                        1: 6086 .. 6091 / #ffffff / -35
                        2: 6092 .. 6109 / #ffffff
                        3: 6110 .. 6116 / #ffffff / -10"
     protein_bind    6124..6140
                     /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)."
                     /note="color: #31849b"
     primer_bind     6148..6164
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     promoter        6185..6203
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
ORIGIN
        1 tgctttctct gaccagcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt
       61 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
      121 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
      181 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
      241 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
      301 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
      361 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
      421 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
      481 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
      541 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
      601 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
      661 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
      721 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
      781 gtcccctcca ccccacagtg gggcaagctt ctgacctctt ctcttcctcc cacagggcct
      841 cgagagatct ggcagcggag agggcagagg aagtcttcta acatgcggtg acgtggagga
      901 gaatcccggc cctaggctcg agacgcgtat gggatcggcc attgaacaag atggattgca
      961 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     1021 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     1081 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     1141 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     1201 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     1261 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     1321 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     1381 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     1441 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     1501 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     1561 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     1621 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     1681 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaggggatcc
     1741 gctgtaagtc tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa
     1801 gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga
     1861 aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcctg ctctttactg
     1921 aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata atttaaacaa
     1981 gcaaaaccaa attaagggcc agctcattcc tcccactcat gatctataga tctatagatc
     2041 tctcgtggga tcattgtttt tctcttgatt cccactttgt ggttctaagt actgtggttt
     2101 ccaaatgtgt cagtttcata gcctgaagaa cgagatcagc agcctctgtt ccacatacac
     2161 ttcattctca gtattgtttt gccaagttct aattccatca gaagctggga gctccaccgc
     2221 ggtggcggcc gctctagaac gagctttact agggacagga ttggtgacag aaaagcccca
     2281 tccttaggcc tcctccttcc tagtctcctg atattgggtc taacccccac ctcctgttag
     2341 gcagattcct tatctggtga cacaccccca tttcctggag ccatctctct ccttgccaga
     2401 acctctaagg tttgcttacg atggagccag agaggatcct gggagggaga gcttggcagg
     2461 gggtgggagg gaaggggggg atgcgtgacc tgcccggttc tcagtggcca ccctgcgcta
     2521 ccctctccca gaacctgagc tgctctgacg cggctgtctg gtgcgtttca ctgatcctgg
     2581 tgctgcagct tccttacact tcccaagagg agaagcagtt tggaaaaaca aaatcagaat
     2641 aagttggtcc tgagttctaa ctttggctct tcacctttct agtccccaat ttatattgtt
     2701 cctccgtgcg tcagttttac ctgtgagata aggccagtag ccagccccgt cctggcaggg
     2761 ctgtggtgag gaggggggtg tccgtgtgga aaactccctt tgtgagaatg gtgcgtccta
     2821 ggtgttcacc aggtcgtggc cgcctctact ccctttctct ttctccatcc ttctttcctt
     2881 aaagagtccc cagtgctatc tgggacatat tcctccgccc agagcagggt cccgcttccc
     2941 taaggccctg ctctgggctt ctgggtttga gtccttggca agcccaggag aggcgctcag
     3001 gcttccctgt cccccttcct cgtccaccat ctcatgcccc tggctctcct gccccttccc
     3061 tacaggggtt cctggctctg ctctaagggc gaattcgcgg ccgctaaatt caattcgccc
     3121 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     3181 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     3241 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctata cgtacggcag
     3301 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt
     3361 gatattattg acacgccggg gcgacggatg gtgatccccc tggccagtgc acgtctgctg
     3421 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc
     3481 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat
     3541 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa
     3601 atgtcaggca tgagattatc aaaaaggatc ttcacctaga tccttttcac gtagaaagcc
     3661 agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat ctggacaagg
     3721 gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta
     3781 gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt
     3841 aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg
     3901 cgcaggggat caagctctga tcaagagaca ggatgaggat cgtttcgcat cttacaattt
     3961 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca
     4021 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4081 tgtatccgct catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4141 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4201 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4261 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4321 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4381 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4441 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4501 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4561 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4621 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4681 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4741 aatactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4801 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4861 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4921 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4981 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5041 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5101 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5161 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5221 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5281 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5341 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5401 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5461 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5521 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5581 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5641 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5701 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5761 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5821 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5881 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5941 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6001 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     6061 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     6121 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     6181 tcagaattaa ccctcactaa agggactagt cctgcaggtt taaacgaatt cgccctt
//
