LOCUS       sequence-321572-        4758 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 4758)
  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 4758)
  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: (326) pAAV SV40-ZsGreen U6-gRNA
FEATURES             Location/Qualifiers
     source          1..4758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        168..525
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        168..359
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(191..211)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      376..511
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      587..596
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             593..1288
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     primer_bind     complement(1328..1345)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1334..1558
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1663..1911
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1663..1683
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1834..1853
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1932..2007
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   2021..2161
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2021..2150
                     /label=AAV2 ITR
     rep_origin      2236..2691
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2323..2342)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2533..2554
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2708..2727)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2827..2849
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2887..2905)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2973..3077
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3078..3146,3147..3938)
                     /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(3296..3315)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4109..4697
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4598..4617
                     /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 gcgttcagtc ctagtgatcc gtaccttctg aggcggaaag
      181 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
      241 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
      301 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
      361 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
      421 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
      481 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct tgattcttct
      541 gacacaacag tctcgctagc ctccagaatt caggtactag tgatccgcca ccatggccca
      601 gtccaagcac ggcctgacca aggagatgac catgaagtac cgcatggagg gctgcgtgga
      661 cggccacaag ttcgtgatca ccggcgaggg catcggctac cccttcaagg gcaagcaggc
      721 catcaacctg tgcgtggtgg agggcggccc cttgcccttc gccgaggaca tcttgtccgc
      781 cgccttcatg tacggcaacc gcgtgttcac cgagtacccc caggacatcg tcgactactt
      841 caagaactcc tgccccgccg gctacacctg ggaccgctcc ttcctgttcg aggacggcgc
      901 cgtgtgcatc tgcaacgccg acatcaccgt gagcgtggag gagaactgca tgtaccacga
      961 gtccaagttc tacggcgtga acttccccgc cgacggcccc gtgatgaaga agatgaccga
     1021 caactgggag ccctcctgcg agaagatcat ccccgtgccc aagcagggca tcttgaaggg
     1081 cgacgtgagc atgtacctgc tgctgaagga cggtggccgc ttgcgctgcc agttcgacac
     1141 cgtgtacaag gccaagtccg tgccccgcaa gatgcccgac tggcacttca tccagcacaa
     1201 gctgacccgc gaggaccgca gcgacgccaa gaaccagaag tggcacctga ccgagcacgc
     1261 catcgcctcc ggctccgcct tgccctagct tccacggtac caagcttaag tttaaaccgc
     1321 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1381 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1441 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     1501 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggat
     1561 tctgaggcgg aaagaaaggt aaccacgtgc ggaccgagcg gatccctagg actgaacgac
     1621 atcttcgaag ctcagaaaat cgaatggcac ggatctggta ccgagggcct atttcccatg
     1681 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
     1741 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     1801 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     1861 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cggagaccac
     1921 ggcaggtctc agttttagta ctctggaaac agaatctact aaaacaaggc aaaatgccgt
     1981 gtttatctcg tcaacttgtt ggcgagattt ttgcggccgc aggaacccct agtgatggag
     2041 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     2101 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     2161 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     2221 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2281 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     2341 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2401 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     2461 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2521 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     2581 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     2641 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     2701 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     2761 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     2821 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     2881 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     2941 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3001 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3061 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3121 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3181 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3241 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3301 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     3361 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3421 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3481 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3541 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3601 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3661 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3721 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3781 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3841 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3901 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3961 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4021 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4081 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4141 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4201 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4261 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4321 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4381 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4441 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4501 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4561 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4621 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4681 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4741 ggccttttgc tcacatgt
//
