LOCUS       sequence-321574-        5504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for Sa gRNA (Bsa1 sites).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5504)
  AUTHORS   Backstrom JR, Sheng J, Wang MC, Bernardo-Colon A, Rex TS
  TITLE     Optimization of S. aureus dCas9 and CRISPRi Elements for a Single 
            Adeno-Associated Virus that Targets an Endogenous Gene.
  JOURNAL   Mol Ther Methods Clin Dev. 2020 Sep 6;19:139-148. doi: 
            10.1016/j.omtm.2020.09.001. eCollection 2020 Dec 11.
  PUBMED    33024795
REFERENCE   2  (bases 1 to 5504)
  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 Ther 
            Methods Clin Dev."; date: "2020-09-6"; volume: "19:139-148. doi"; 
            pages: " 10.1016/j.omtm.2020.09.001. eCollection 2020 Dec 11"
COMMENT     Sequence Label: (401) pAAV CMV-ZsGreen U6-gRNA
FEATURES             Location/Qualifiers
     source          1..5504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        206..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     664..684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     710..734
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          862..1240
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     regulatory      1333..1342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1339..2034
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     primer_bind     complement(2074..2091)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2080..2304
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2409..2657
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2409..2429
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2580..2599
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2678..2753
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   2767..2907
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2767..2896
                     /label=AAV2 ITR
     rep_origin      2982..3437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3069..3088)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3279..3300
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3454..3473)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3573..3595
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3633..3651)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3719..3823
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3824..3892,3893..4684)
                     /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(4042..4061)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4855..5443
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5344..5363
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtctagtt attaatagta atcaattacg gggtcattag
      181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      541 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      601 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      661 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag
      721 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
      781 gggaccgatc cagcctccgc ggattcgaat cccggccggg aacggtgcat tggaacgcgg
      841 attccccgtg ccaagagtga cgtaagtacc gcctatagag tctataggcc cacaaaaaat
      901 gctttcttct tttaatatac ttttttgttt atcttatttc taatactttc cctaatctct
      961 ttctttcagg gcaataatga tacaatgtat catgcctctt tgcaccattc taaagaataa
     1021 cagtgataat ttctgggtta aggcaatagc aatatttctg catataaata tttctgcata
     1081 taaattgtaa ctgatgtaag aggtttcata ttgctaatag cagctacaat ccagctacca
     1141 ttctgctttt attttatggt tgggataagg ctggattatt ctgagtccaa gctaggccct
     1201 tttgctaatc atgttcatac ctcttatctt cctcccacag ctcctgggca acgtgctggt
     1261 ctgtgtgctg gcccatcact ttggcaaaga attgggattc gaacatcgat tgaattcagg
     1321 tactagtgat ccgccaccat ggcccagtcc aagcacggcc tgaccaagga gatgaccatg
     1381 aagtaccgca tggagggctg cgtggacggc cacaagttcg tgatcaccgg cgagggcatc
     1441 ggctacccct tcaagggcaa gcaggccatc aacctgtgcg tggtggaggg cggccccttg
     1501 cccttcgccg aggacatctt gtccgccgcc ttcatgtacg gcaaccgcgt gttcaccgag
     1561 tacccccagg acatcgtcga ctacttcaag aactcctgcc ccgccggcta cacctgggac
     1621 cgctccttcc tgttcgagga cggcgccgtg tgcatctgca acgccgacat caccgtgagc
     1681 gtggaggaga actgcatgta ccacgagtcc aagttctacg gcgtgaactt ccccgccgac
     1741 ggccccgtga tgaagaagat gaccgacaac tgggagccct cctgcgagaa gatcatcccc
     1801 gtgcccaagc agggcatctt gaagggcgac gtgagcatgt acctgctgct gaaggacggt
     1861 ggccgcttgc gctgccagtt cgacaccgtg tacaaggcca agtccgtgcc ccgcaagatg
     1921 cccgactggc acttcatcca gcacaagctg acccgcgagg accgcagcga cgccaagaac
     1981 cagaagtggc acctgaccga gcacgccatc gcctccggct ccgccttgcc ctagcttcca
     2041 cggtaccaag cttaagttta aaccgctgat cagcctcgac tgtgccttct agttgccagc
     2101 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     2161 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     2221 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     2281 ctggggatgc ggtgggctct atggattctg aggcggaaag aaaggtaacc acgtgcggac
     2341 cgagcggatc cctaggactg aacgacatct tcgaagctca gaaaatcgaa tggcacggat
     2401 ctggtaccga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     2461 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     2521 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     2581 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     2641 ggaaaggacg aaacaccgga gaccacggca ggtctcagtt ttagtactct ggaaacagaa
     2701 tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg agatttttgc
     2761 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     2821 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     2881 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     2941 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     3001 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     3061 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3121 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3181 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3241 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3301 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     3361 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     3421 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3481 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3541 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3601 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     3661 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     3721 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3781 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     3841 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     3901 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     3961 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4021 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4081 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4141 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4201 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4261 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     4321 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     4381 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     4441 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     4501 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     4561 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     4621 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     4681 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     4741 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4801 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4861 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4921 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4981 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5041 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5101 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5161 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5221 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5281 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5341 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5401 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5461 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
