LOCUS       sequence-242711-        8195 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DD-AcrIIA4 fusion for Shield1-mediated AcrIIA4 control without sgRNA
            following mU6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8195)
  AUTHORS   Nakamura M, Srinivasan P, Chavez M, Carter MA, Dominguez AA, La 
            Russa M, Lau MB, Abbott TR, Xu X, Zhao D, Gao Y, Kipniss NH, Smolke 
            CD, Bondy-Denomy J, Qi LS
  TITLE     Anti-CRISPR-mediated control of gene editing and synthetic circuits 
            in eukaryotic cells.
  JOURNAL   Nat Commun. 2019 Jan 14;10(1):194. doi: 10.1038/s41467-018-08158-x.
  PUBMED    30643127
REFERENCE   2  (bases 1 to 8195)
  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: "Nat 
            Commun."; date: "2019-01-14"; volume: "10(1):194. doi"; pages: " 
            10.1038/s41467-018-08158-x"
COMMENT     Sequence Label: pSLQ7780 mU6:- || CMV: mCherry~P2A-DD-AcrIIA4
FEATURES             Location/Qualifiers
     source          1..8195
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             834..1014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1061..1186
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1683..1916
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2101..2145
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2443..2560
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2813..2846)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2850..2870
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        3063..3366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3367..3570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3613..4317
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3755..3773)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4023..4042
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4327..4383
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    complement(4994..5027)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5083..5671
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5136..5156)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5554..5565)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5674..5690)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5675..5691)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6200..6380
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6442..7030)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6522..6541)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7201..7992,7993..8061))
                     /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     7824..7843
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8062..8166)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     1021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     1081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     1201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     1261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     1321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     1381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     1441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     1501 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat
     1561 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1621 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1681 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1741 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1801 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1861 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1921 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1981 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     2041 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     2101 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     2161 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     2221 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2281 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2341 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2401 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2461 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2521 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2581 cagagatcca gtttggttag taccgggccc gctctagaga tccgacgcgc catctctagg
     2641 cccgcgccgg ccccctcgca cggacttgtg ggagaagctc ggctactccc ctgccccggt
     2701 taatttgcat ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat
     2761 aatctgttct ttttaatact agctacattt tacatgatag gcttggattt ctataacttc
     2821 gtatagcata cattatacga agttataaac agcacaaaag gaaactcacc ctaactgtaa
     2881 agtaattgtg tgttttgaga ctataagtat cccttggaga accaccttgt tggcgcgcca
     2941 tcagtctcga gtactaggat ccattaggcg gccgcgtgga taaccgtatt accgccatgc
     3001 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3061 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     3121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     3301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     3361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     3421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     3481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     3541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagccc
     3601 accatgcccg gggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
     3661 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     3721 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     3781 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     3841 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     3901 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     3961 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     4021 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     4081 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     4141 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     4201 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     4261 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagggt
     4321 tctggagcta ctaacttctc cctgctgaaa caggcaggag acgtggagga gaaccctgga
     4381 ccaggagtgc aagtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc
     4441 cagacctgtg tggtgcacta caccgggatg cttgaagatg gaaagaaagt cgattcctcc
     4501 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg
     4561 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat
     4621 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc
     4681 gatgtggagc ttctaaaacc tgaaggatct ggaaacatca acgatctgat ccgggagatc
     4741 aagaacaagg actacaccgt gaagctgagc ggcaccgata gcaacagcat cacccagctg
     4801 attatccgcg tgaacaacga cggcaacgag tacgtgatct ccgagagcga gaacgagagc
     4861 atcgtggaga agttcatcag cgccttcaag aacggttgga accaggagta cgaggacgag
     4921 gaggagttct acaacgacat gcagaccatc accctgaaga gcgagctgaa ctaagaattc
     4981 gtcgagggac ctaataactt cgtatagcat acattatacg aagttataca tgtttaaggg
     5041 ttccggttcc actaggtaca attcgatatc aagcttatcg ataatcaacc tctggattac
     5101 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     5161 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     5221 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     5281 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     5341 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     5401 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     5461 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     5521 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     5581 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     5641 agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacctcg atcgagacct
     5701 agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg attgtgcctg
     5761 gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg tacctttaag
     5821 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     5881 ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac
     5941 acaaggctac ttccctgatt ggcagaacta cacaccaggg ccagggatca gatatccact
     6001 gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag aagaagccaa
     6061 tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg atgacccgga
     6121 gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca tggcccgaga
     6181 gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     6241 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     6301 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     6361 agtgtggaaa atctctagca gcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6421 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6481 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6541 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6601 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6661 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6721 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6781 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6841 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6901 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6961 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     7021 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     7081 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     7141 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     7201 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7261 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7321 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     7381 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7441 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7501 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7561 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7621 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7681 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7741 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7801 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7861 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7921 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7981 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     8041 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     8101 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     8161 tccgcgcaca tttccccgaa aagtgccacc tgacg
//
