LOCUS       sequence-208521-        5338 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pX552-CMV-GFP. AAV plasmid for sgRNA cloning, with GFP fusion 
            facilitating FACS sorting of cells. Replaced the hSyn promoter in 
            pX552 from Zhang lab with a CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5338)
  TITLE     Alex Hewitt Lab plasmids
REFERENCE   2  (bases 1 to 5338)
  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"
COMMENT     Sequence Label: pX552-GFP
FEATURES             Location/Qualifiers
     source          1..5338
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   8..137
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        152..400
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     152..172
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     323..342
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        419..494
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        520..805
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        806..1009
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     960..980
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1053..1062
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1059..1061,1062..1064,1065..1775)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1059..1061,1062..1064,1065..1775)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1104..1125)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1365..1384)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1712..1733
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2092..2568
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2291..2310)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2608..2748
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2608..2737
                     /label=AAV2 ITR
     rep_origin      2823..3278
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2910..2929)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3120..3141
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3295..3314)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3414..3436
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3474..3492)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3560..3664
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3665..3733,3734..4525)
                     /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(3883..3902)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4696..5284
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5185..5204
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc
       61 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
      121 catcactagg ggttcctgcg gccgcacgcg tgagggccta tttcccatga ttccttcata
      181 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
      241 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      301 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      361 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggaagagcga gctcttctgt
      421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      481 caccgagtcg gtgctttttt tctagactgc agagggcccc gttacataac ttacggtaaa
      541 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat
      601 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      661 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      721 cgcctggcat tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      781 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      841 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      901 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      961 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa
     1021 ccgtcagagg taccggatcc tctagagtcg acgccaccat ggtgagcaag ggcgaggagc
     1081 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1141 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1201 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1261 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1321 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1381 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1441 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     1501 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     1561 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1621 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     1681 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1741 ccgggatcac tctcggcatg gacgagctgt acaagtccgg actcagatct cgagaggagg
     1801 aggaggagac agacagcagg atgccccacc tcgacagccc cggcagctcc cagccgagac
     1861 gctccttcct ctcaagggtg atcagggcag cgctaccgtt gcagctgctt ctgctgctgc
     1921 tgctgctcct ggcctgcctg ctacctgcct ctgaagatga ctacagctgc acccaggcca
     1981 acaactttgc ccgatccttc taccccatgc tgcggtacac caacgggcca cctcccacct
     2041 aggaattcga tatcaagctt atcgataccg agcgctgctc gagagatcta cgggtggcat
     2101 ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc
     2161 agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat
     2221 attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg
     2281 gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
     2341 atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc
     2401 caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc
     2461 atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc
     2521 aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag
     2581 gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct
     2641 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     2701 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     2761 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     2821 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2881 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2941 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3001 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     3061 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3121 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     3181 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     3241 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     3301 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     3361 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3421 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     3481 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     3541 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3601 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3661 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3721 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3781 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3841 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3901 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3961 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4021 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4081 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4141 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4201 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4261 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4321 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4381 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4441 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4501 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4561 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4621 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4681 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4741 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4801 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     4861 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4921 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4981 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5041 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5101 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5161 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5221 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5281 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgct
//
