LOCUS       sequence-384998-        8165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1 Integratable mCherry Reporter for synZiFTR ZF3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8165)
  AUTHORS   Li HS, Israni DV, Gagnon KA, Gan KA, Raymond MH, Sander JD, Roybal 
            KT, Joung JK, Wong WW, Khalil AS
  TITLE     Multidimensional control of therapeutic human cell function with 
            synthetic gene circuits.
  JOURNAL   Science. 2022 Dec 16;378(6625):1227-1234. doi: 
            10.1126/science.ade0156. Epub 2022 Dec 15.
  PUBMED    36520914
REFERENCE   2  (bases 1 to 8165)
  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"; doi: 
            "10.1126/science.ade0156"; journalName: "Science"; date: 
            "2022-12-16- 16"; volume: "378"; issue: "6625"; pages: "1227-1234"
COMMENT     Sequence Label: pMN-011
FEATURES             Location/Qualifiers
     source          1..8165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    127..656
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     1094..1118
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1141..1150
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1147..1857
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWQASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1292..1310)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1560..1579
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1943..1962)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2008..2063
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2062..2081)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    2409..2633
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2698..3197
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2733..2756)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3130..3149
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3221..3817
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3221..3240)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3717..3737
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3827..3883
                     /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"
     CDS             3884..4585
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4703..4824
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    4965..5466
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(5668..5684)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5668..5684)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5681..5703)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5692..5708
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5716..5722,5723..5740,5741..5746))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5761..5782
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5841..6036
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5887..6022
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6042..6176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6244..6261)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6415..7003)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6495..6514)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7174..7965,7966..8034))
                     /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     7797..7816
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8035..8139)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agtgaagggg caggagagcc aggggcatga gatggtggac
       61 gaggaagggg gacagggaag cctgagcgcc tctcctgggc ttgccaagga ctcaaaccca
      121 gaagcccaga gcagggcctt agggaagcgg gaccctgctc tgggcggagg aatatgtccc
      181 agatagcact ggggactctt taaggaaaga aggatggaga aagagaaagg gagtagaggc
      241 ggccacgacc tggtgaacac ctaggacgca ccattctcac aaagggagtt ttccacacgg
      301 acacccccct cctcaccaca gccctgccag gacggggctg gctactggcc ttatctcaca
      361 ggtaaaactg acgcacggag gaacaatata aattggggac tagaaaggtg aagagccaaa
      421 gttagaactc aggaccaact tattctgatt ttgtttttcc aaactgcttc tcctcttggg
      481 aagtgtaagg aagctgcagc accaggatca gtgaaacgca ccagacagcc gcgtcagagc
      541 agctcaggtt ctgggagagg gtagcgcagg gtggccactg agaaccgggc aggtcacgca
      601 tccccccctt ccctcccacc ccctgccaag ctctccctcc caggatcctc tctggctcca
      661 tcgtaagcaa accttagagg ttctggcaag gagagagatg gctccaggaa atgggggtgt
      721 gtcaccagat aaggaatctg cctaacagga ggtgggggtt agacccaata tcaggagact
      781 aggaaggagg aggcctaagg atggggcttt tctgtcacca atccgccggc gaattcgtcg
      841 accgggtttc gtaacaatcg catgaggatt cgcaacgcct tagacgtcga agtagccgta
      901 gtcccgtctc agtaaaggta gacgtcgaag tagccgtaga atcggactgc cttcgtaaga
      961 cgtcgaagta gccgtaggta tcagtcgcct cggaaagacg tcgaagtagc cgtagattcg
     1021 taagaggctc actctccctt acacggagtg gataactagt taggcgtgta cggtgggagg
     1081 cctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggaacgcg taccggtgtc
     1141 gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     1201 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1261 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     1321 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     1381 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     1441 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     1501 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1561 cccgtaatgc agaagaagac catgggctgg caggcctcct ccgagcggat gtaccccgag
     1621 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     1681 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     1741 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     1801 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaagcg
     1861 gccgcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     1921 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     1981 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     2041 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     2101 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     2161 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     2221 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     2281 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     2341 tctcctgact actcccagtc atagctgtcc ctcttctctt atgaagatcc ctcgacctgc
     2401 agggtaccct gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2461 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2521 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2581 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2641 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcgtaccggg
     2701 taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca
     2761 cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc
     2821 aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga
     2881 agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc
     2941 tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg
     3001 gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg
     3061 ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg
     3121 gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct
     3181 catctccggg cctttcgata tctcgcacag tgccgccacc atgaccgagt acaagcccac
     3241 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     3301 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     3361 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     3421 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     3481 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3541 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3601 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3661 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     3721 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     3781 accgcgcacc tggtgcatga cccgcaagcc cggtgccgga agcggagcta ctaacttcag
     3841 cctgctgaag caggctggag acgtggagga gaaccctgga cctatgagcg agctgattaa
     3901 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg
     3961 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt
     4021 cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag
     4081 caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga
     4141 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
     4201 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
     4261 cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac
     4321 gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg
     4381 cgggagccat ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa
     4441 cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa
     4501 caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag
     4561 caaactgggg cacaagctta attaatctag ataactgatc ataatcagcc ataccacatt
     4621 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     4681 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     4741 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4801 gtccaaactc atcaatgtat cttaggcgcg ccctgtccct agtggcccca ctgtggggtg
     4861 gaggggacag ataaaagtac ccagaaccag agccacatta accggccctg ggaatataag
     4921 gtggtcccag ctcggggaca caggatccct ggaggcagca aacatgctgt cctgaagtgg
     4981 acataggggc ccgggttgga ggaagaagac tagctgagct ctcggacccc tggaagatgc
     5041 catgacaggg ggctggaaga gctagcacag actagagagg taaggggggt aggggagctg
     5101 cccaaatgaa aggagtgaga ggtgacccga atccacagga gaacggggtg tccaggcaaa
     5161 gaaagcaaga ggatggagag gtggctaaag ccagggagac ggggtacttt ggggttgtcc
     5221 agaaaaacgg tgatgatgca ggcctacaag aaggggaggc gggacgcaag ggagacatcc
     5281 gtcggagaag gccatcctaa gaaacgagag atggcacagg ccccagaagg agaaggaaaa
     5341 gggaacccag cgagtgaaga cggcatgggg ttgggtgagg gaggagagat gcccggagag
     5401 gacccagaca cggggaggat ccgctcagag gacatcacgt ggtgcagcgc cgagaaggaa
     5461 gtgctccgga aagagcatcc ttgggcagca acacagcaga gagcaagggg aagagggagt
     5521 ggaggaagac ggaacctgaa ggaggcggca gggaaggatc tgggccagcc gtagaggtga
     5581 cccaggccac aagctgcaga cagaaagcgg cacaggccca ggggagagaa tgccctcgac
     5641 ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     5701 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     5761 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5821 cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     5881 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     5941 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     6001 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt
     6061 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     6121 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct
     6181 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     6241 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6301 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     6361 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6421 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6481 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6541 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6601 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6661 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6721 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6781 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6841 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6901 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6961 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     7021 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     7081 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     7141 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7201 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7261 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7321 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7381 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7441 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7501 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7561 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7621 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7681 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7741 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7801 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7861 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7921 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7981 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     8041 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     8101 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     8161 acctg
//
