LOCUS       sequence-242710-        8296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DD-AcrIIA4 fusion for Shield1-mediated AcrIIA4 control with sgRNA 
            driving TRE3G activation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8296)
  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 8296)
  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: pSLQ7748 mU6: Spy sgTET || CMV: mCherry~P2A-DD-A4
FEATURES             Location/Qualifiers
     source          1..8296
                     /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        3164..3467
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3468..3671
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3714..4418
                     /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(3856..3874)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4124..4143
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4428..4484
                     /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(5095..5128)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5184..5772
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5237..5257)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5655..5666)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5775..5791)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5776..5792)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6301..6481
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6543..7131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6623..6642)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7302..8093,8094..8162))
                     /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     7925..7944
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8163..8267)
                     /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 tggtacgttc
     2941 tctatcactg atagtttaag agctatgctg gaaacagcat agcaagttta aataaggcta
     3001 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctcg agtactagga
     3061 tccattaggc ggccgcgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     3121 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     3181 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     3241 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     3301 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     3361 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     3421 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     3481 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     3541 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     3601 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     3661 taagcagagc tggtttagtg aaccgtcaga tccgctagcc caccatgccc ggggtgagca
     3721 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     3781 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     3841 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     3901 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     3961 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     4021 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     4081 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     4141 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     4201 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     4261 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     4321 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     4381 gccgccactc caccggcggc atggacgagc tgtacaaggg ttctggagct actaacttct
     4441 ccctgctgaa acaggcagga gacgtggagg agaaccctgg accaggagtg caagtggaaa
     4501 ccatctcccc aggagacggg cgcaccttcc ccaagcgcgg ccagacctgt gtggtgcact
     4561 acaccgggat gcttgaagat ggaaagaaag tcgattcctc ccgggacaga aacaagccct
     4621 ttaagtttat gctaggcaag caggaggtga tccgaggctg ggaagaaggg gttgcccaga
     4681 tgagtgtggg tcagagagcc aaactgacta tatctccaga ttatgcctat ggtgccactg
     4741 ggcacccagg catcatccca ccacatgcca ctctcgtctt cgatgtggag cttctaaaac
     4801 ctgaaggatc tggaaacatc aacgatctga tccgggagat caagaacaag gactacaccg
     4861 tgaagctgag cggcaccgat agcaacagca tcacccagct gattatccgc gtgaacaacg
     4921 acggcaacga gtacgtgatc tccgagagcg agaacgagag catcgtggag aagttcatca
     4981 gcgccttcaa gaacggttgg aaccaggagt acgaggacga ggaggagttc tacaacgaca
     5041 tgcagaccat caccctgaag agcgagctga actaagaatt cgtcgaggga cctaataact
     5101 tcgtatagca tacattatac gaagttatac atgtttaagg gttccggttc cactaggtac
     5161 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     5221 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     5281 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     5341 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     5401 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     5461 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     5521 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     5581 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     5641 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     5701 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     5761 ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc
     5821 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag
     5881 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca
     5941 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     6001 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
     6061 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac
     6121 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc
     6181 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg
     6241 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact
     6301 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     6361 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     6421 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     6481 agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6541 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6601 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6661 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6721 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6781 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6841 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6901 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6961 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     7021 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     7081 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     7141 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7201 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7261 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7321 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7381 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7441 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7501 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7561 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7621 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7681 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7741 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7801 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7861 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7921 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7981 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     8041 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     8101 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     8161 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     8221 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8281 aaagtgccac ctgacg
//
