LOCUS       sequence-353737-        6835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter_ZIFUAS_minCMVpromoter_GFP_AAVS1_Puromycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6835)
  AUTHORS   Staller MV, Ramirez E, Kotha SR, Holehouse AS, Pappu RV, Cohen BA
  TITLE     Directed mutational scanning reveals a balance between acidic and 
            hydrophobic residues in strong human activation domains.
  JOURNAL   Cell Syst. 2022 Apr 20;13(4):334-345.e5. doi: 
            10.1016/j.cels.2022.01.002. Epub 2022 Feb 3.
  PUBMED    35120642
REFERENCE   2  (bases 1 to 6835)
  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: 
            "10.1016/j.cels.2022.01.002"; journalName: "Cell Syst"; date: 
            "2022-04-20- 20"; volume: "13"; issue: "4"; pages: "334-345.e5"
COMMENT     Sequence Label: pMVS184
FEATURES             Location/Qualifiers
     source          1..6835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(60..515)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(197..218)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     409..428
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     642..664
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     656..673
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     657..673
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     683..702
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        683..701
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    716..1519
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    1520..1545
                     /label=SA
                     /note="splice acceptor site"
     CDS             1569..1622
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1632..2231
                     /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(1632..1651)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2128..2148
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2379..2396)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2385..2496
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2837..2861
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     polyA_signal    3616..3737
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3627..3646)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3681..3700
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    3838..4674
                     /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     complement(4704..4724)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4704..4722)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4743..4759)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4743..4759)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4756..4778)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4767..4783
                     /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(4791..4797,4798..4815,4816..4821))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4836..4857
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4974..4991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5145..5733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5225..5244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5904..6695,6696..6764))
                     /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     6527..6546
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6765..6835,1..34))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
       61 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
      121 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
      181 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
      241 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
      301 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
      361 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
      421 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
      481 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
      541 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
      601 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
      661 aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtacctgctt
      721 tctctgacca gcattctctc ccctgggcct gtgccgcttt ctgtctgcag cttgtggcct
      781 gggtcacctc tacggctggc ccagatcctt ccctgccgcc tccttcaggt tccgtcttcc
      841 tccactccct cttccccttg ctctctgctg tgttgctgcc caaggatgct ctttccggag
      901 cacttccttc tcggcgctgc accacgtgat gtcctctgag cggatcctcc ccgtgtctgg
      961 gtcctctccg ggcatctctc ctccctcacc caaccccatg ccgtcttcac tcgctgggtt
     1021 cccttttcct tctccttctg gggcctgtgc catctctcgt ttcttaggat ggccttctcc
     1081 gacggatgtc tcccttgcgt cccgcctccc cttcttgtag gcctgcatca tcaccgtttt
     1141 tctggacaac cccaaagtac cccgtctccc tggctttagc cacctctcca tcctcttgct
     1201 ttctttgcct ggacaccccg ttctcctgtg gattcgggtc acctctcact cctttcattt
     1261 gggcagctcc cctacccccc ttacctctct agtctgtgct agctcttcca gccccctgtc
     1321 atggcatctt ccaggggtcc gagagctcag ctagtcttct tcctccaacc cgggccccta
     1381 tgtccacttc aggacagcat gtttgctgcc tccagggatc ctgtgtcccc gagctgggac
     1441 caccttatat tcccagggcc ggttaatgtg gctctggttc tgggtacttt tatctgtccc
     1501 ctccacccca cagtggggcc tgacctcttc tcttcctccc acagggcctc gagagatctg
     1561 gcagcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag aatcccggcc
     1621 ctaggctcga aatgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     1681 cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     1741 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     1801 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     1861 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     1921 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     1981 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
     2041 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     2101 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     2161 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     2221 ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc
     2281 atggctccga ccgaagccac ccggggcggc cccgccgacc ccgcacccgc ccccgaggcc
     2341 caccgactct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     2401 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2461 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2521 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcaa
     2581 ccggtcgggt ttcgtaacaa tcgcatgagg attcgcaacg ccttcgtcga agtcgaagtc
     2641 gacctcccgt ctcagtaaag gtcgtcgaag tcgaagtcga ccaatcggac tgccttcgta
     2701 cgtcgaagtc gaagtcgacc gtatcagtcg cctcggaacg tcgaagtcga agtcgaccat
     2761 tcgtaagagg ctcactctcc cttacacgga gtggataact agttaggcgt gtacggtggg
     2821 aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaac gcgtaccggt
     2881 gtcgccacca tgagtaaagg cgaagagctc tttacaggtg tggtgcccat ccttgtcgaa
     2941 cttgatgggg acgtgaatgg ccataaattt tctgtaagcg gtgaaggaga gggcgatgca
     3001 acttacggta agctcacact caagtttatc tgcaccacag gtaaactgcc cgtgccctgg
     3061 ccgaccctgg tcacgacgtt cggttacggt gtgcaatgct ttgcgcggta tccagatcat
     3121 atgaaacaac atgacttttt taagtccgcg atgccggagg ggtatgtcca ggaaaggact
     3181 atcttcttca aggatgacgg taactataag acacgggccg aggttaaatt cgagggtgac
     3241 acccttgtca accgcatcga attgaagggg atagacttca aggaagatgg gaatatactg
     3301 gggcacaaac tggaatataa ttataatagt cacaacgttt atattatggc ggataagcaa
     3361 aagaacggaa tcaaggtgaa cttcaaaata aggcacaaca tagaagacgg atccgtacaa
     3421 ttggccgatc actatcaaca aaatacgcca ataggggacg gcccagtcct tctccccgac
     3481 aatcattatc tgtctactca aagtgcgctc tcaaaagacc caaacgagaa aagagatcac
     3541 atggttctgc tggaatttgt gacagccgct ggaatcacac acggaatgga cgaactttac
     3601 aaataacaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     3661 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     3721 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     3781 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtagc ggccgctact
     3841 agggacagga ttggtgacag aaaagcccca tccttaggcc tcctccttcc tagtctcctg
     3901 atattgggtc taacccccac ctcctgttag gcagattcct tatctggtga cacaccccca
     3961 tttcctggag ccatctctct ccttgccaga acctctaagg tttgcttacg atggagccag
     4021 agaggatcct gggagggaga gcttggcagg gggtgggagg gaaggggggg atgcgtgacc
     4081 tgcccggttc tcagtggcca ccctgcgcta ccctctccca gaacctgagc tgctctgacg
     4141 cggctgtctg gtgcgtttca ctgatcctgg tgctgcagct tccttacact tcccaagagg
     4201 agaagcagtt tggaaaaaca aaatcagaat aagttggtcc tgagttctaa ctttggctct
     4261 tcacctttct agtccccaat ttatattgtt cctccgtgcg tcagttttac ctgtgagata
     4321 aggccagtag ccagccccgt cctggcaggg ctgtggtgag gaggggggtg tccgtgtgga
     4381 aaactccctt tgtgagaatg gtgcgtccta ggtgttcacc aggtcgtggc cgcctctact
     4441 ccctttctct ttctccatcc ttctttcctt aaagagtccc cagtgctatc tgggacatat
     4501 tcctccgccc agagcagggt cccgcttccc taaggccctg ctctgggctt ctgggtttga
     4561 gtccttggca agcccaggag aggcgctcag gcttccctgt cccccttcct cgtccaccat
     4621 ctcatgcccc tggctctcct gccccttccc tacaggggtt cctggctctg ctctccaccg
     4681 cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     4741 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4801 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4861 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4921 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4981 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5041 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5101 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5161 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5221 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5281 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5341 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5401 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5461 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5521 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5581 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5641 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5701 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5761 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5821 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5881 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5941 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6001 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6061 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6121 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6181 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6241 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6301 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6361 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6421 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6481 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6541 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6601 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6661 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6721 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6781 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaat
//
