LOCUS       sequence-315765-        5507 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 5507)
  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 5507)
  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..5507
                     /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        210..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        514..716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     667..687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     713..737
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          865..1243
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     regulatory      1336..1345
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1342..2037
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     primer_bind     complement(2077..2094)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2083..2307
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2412..2660
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2412..2432
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2583..2602
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2681..2756
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   2770..2910
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2770..2899
                     /label=AAV2 ITR
     rep_origin      2985..3440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3072..3091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3282..3303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3457..3476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3576..3598
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3636..3654)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3722..3826
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3827..3895,3896..4687)
                     /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(4045..4064)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4858..5446
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5347..5366
                     /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 gcgtggagct agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgtcaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      541 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      601 gggagtttgt tttgcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      661 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt
      721 tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac
      781 accgggaccg atccagcctc cgcggattcg aatcccggcc gggaacggtg cattggaacg
      841 cggattcccc gtgccaagag tgacgtaagt accgcctata gagtctatag gcccacaaaa
      901 aatgctttct tcttttaata tacttttttg tttatcttat ttctaatact ttccctaatc
      961 tctttctttc agggcaataa tgatacaatg tatcatgcct ctttgcacca ttctaaagaa
     1021 taacagtgat aatttctggg ttaaggcaat agcaatattt ctgcatataa atatttctgc
     1081 atataaattg taactgatgt aagaggtttc atattgctaa tagcagctac aatccagcta
     1141 ccattctgct tttattttat ggttgggata aggctggatt attctgagtc caagctaggc
     1201 ccttttgcta atcatgttca tacctcttat cttcctccca cagctcctgg gcaacgtgct
     1261 ggtctgtgtg ctggcccatc actttggcaa agaattggga ttcgaacatc gattgaattc
     1321 aggtactagt gatccgccac catggcccag tccaagcacg gcctgaccaa ggagatgacc
     1381 atgaagtacc gcatggaggg ctgcgtggac ggccacaagt tcgtgatcac cggcgagggc
     1441 atcggctacc ccttcaaggg caagcaggcc atcaacctgt gcgtggtgga gggcggcccc
     1501 ttgcccttcg ccgaggacat cttgtccgcc gccttcatgt acggcaaccg cgtgttcacc
     1561 gagtaccccc aggacatcgt cgactacttc aagaactcct gccccgccgg ctacacctgg
     1621 gaccgctcct tcctgttcga ggacggcgcc gtgtgcatct gcaacgccga catcaccgtg
     1681 agcgtggagg agaactgcat gtaccacgag tccaagttct acggcgtgaa cttccccgcc
     1741 gacggccccg tgatgaagaa gatgaccgac aactgggagc cctcctgcga gaagatcatc
     1801 cccgtgccca agcagggcat cttgaagggc gacgtgagca tgtacctgct gctgaaggac
     1861 ggtggccgct tgcgctgcca gttcgacacc gtgtacaagg ccaagtccgt gccccgcaag
     1921 atgcccgact ggcacttcat ccagcacaag ctgacccgcg aggaccgcag cgacgccaag
     1981 aaccagaagt ggcacctgac cgagcacgcc atcgcctccg gctccgcctt gccctagctt
     2041 ccacggtacc aagcttaagt ttaaaccgct gatcagcctc gactgtgcct tctagttgcc
     2101 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2161 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2221 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2281 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaaggta accacgtgcg
     2341 gaccgagcgg atccctagga ctgaacgaca tcttcgaagc tcagaaaatc gaatggcacg
     2401 gatctggtac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     2461 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     2521 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     2581 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     2641 tgtggaaagg acgaaacacc ggagaccacg gcaggtctca gttttagtac tctggaaaca
     2701 gaatctacta aaacaaggca aaatgccgtg tttatctcgt caacttgttg gcgagatttt
     2761 tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     2821 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     2881 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     2941 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     3001 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3061 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3121 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3181 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3241 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3301 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     3361 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3421 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     3481 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3541 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3601 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     3661 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     3721 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3781 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3841 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3901 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3961 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4021 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4081 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4141 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4201 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4261 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4321 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4381 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4441 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4501 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     4561 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4621 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4681 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4741 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4801 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4861 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4921 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4981 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     5041 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5101 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5161 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5221 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5281 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5341 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5401 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5461 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
