LOCUS       sequence-414330-        8041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of uMASS_LF (loss-of-function) in mammalian cells under 
            CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8041)
  AUTHORS   Rappleye M, Wait SJ, Lee JD, Siebart JC, Torp L, Smith N, Muster J, 
            Matreyek KA, Fowler DM, Berndt A
  TITLE     Optogenetic Microwell Array Screening System: A High-Throughput 
            Engineering Platform for Genetically Encoded Fluorescent Indicators.
  JOURNAL   ACS Sens. 2023 Nov 24;8(11):4233-4244. doi: 
            10.1021/acssensors.3c01573. Epub 2023 Nov 13.
  PUBMED    37956352
REFERENCE   2  (bases 1 to 8041)
  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: 
            "10.1021/acssensors.3c01573"; journalName: "ACS Sens"; date: 
            "2023-11-24- 24"; volume: "8"; issue: "11"; pages: "4233-4244"
COMMENT     Sequence Label: pCAG--uMASS_LF
FEATURES             Location/Qualifiers
     source          1..8041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    24..35
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     enhancer        39..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        420..694
                     /label=chicken beta-actin promoter
     intron          695..1711
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1635..1657
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1719..1738
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2860..2881
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2987..3008)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3248..3267)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     intron          3932..4064
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4047..4066
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    4073..4661
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4126..4146)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4544..4555)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4688..4705)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4694..4805
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4901..4921)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4901..4919)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4940..4956)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5062..5081)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5127..5182
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5181..5200)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5543..5559)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5567..5583
                     /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(5591..5597,5598..5615,5616..5621))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5636..5657
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5716..5911
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5762..5897
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5917..6051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5954..5973)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6008..6027
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6119..6136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6290..6878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6370..6389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7049..7840,7841..7909))
                     /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     7672..7691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7910..8014)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agagtcatcc tcatcgtcga cattgattat tgactagtta
       61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      241 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      421 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
      481 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
      541 ggcgccgggg gggggggggg gggggggggg gcggggcggg gcgaggcgga gaggtgcggc
      601 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
      661 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
      721 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
      781 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta
      841 atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc
      901 tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc
      961 gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc
     1021 gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg
     1081 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     1141 gggcgcgtcg gtcgggctgc aacgccccct gcacccccct ccccgagttg ctgagcacgg
     1201 cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg
     1261 ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc
     1321 gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     1381 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     1441 tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg
     1501 gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg
     1561 tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg
     1621 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
     1681 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
     1741 gtctcatcat tttggcaaag aattcggtac cgccaccatg aagacgatca tcgccctgag
     1801 ctacatcttc tgcctggtgt tcgccgacta caaggacgat gatgacgcca tggacagcag
     1861 caccggccca gggaacacca gcgactgctc agacccctta gctcaggcaa gttgctcccc
     1921 agcacctggc tcctggctca acttgtccca cgttgatggc aaccagtccg atccatgcgg
     1981 tctgaaccgc accgggcttg gcgggaacga cagcctgtgc cctcagaccg gcagcccttc
     2041 catggtcaca gccattacca tcatggccct ctactctatc gtgtgtgtag tgggcctctt
     2101 cggaaacttc ctggtcatgt atgtgattgt aagatacacc aaaatgaaga ctgccaccaa
     2161 catctacatt ttcaaccttg ctctggcaga cgccttagcg accagtacac tgccctttca
     2221 gagtgtcaac tacctgatgg gaacatggcc cttcggaacc atcctctgca agatcgtgat
     2281 ctcaatagga tactacaaca tgttcaccag catattcacc ctctgcacca tgagcgtgga
     2341 ccgctacatt gctgtctgcc acccagtcaa agccctggat ttccgtaccc cccgaaatgc
     2401 caaaatcgtc aacgtctgca actggatcct ctcttctgcc atcggtctgc ctgtaatgtt
     2461 catggcaacc acgaaataca ggcaggggtc catagattgc accctcacgt tctcccaccc
     2521 aacctggtac tgggagaacc tgctcaaaat ctgtgtcttt atcttcgctt tcatcatgcc
     2581 gatcctcatc atcactgtgt gttacggcct gatgatctta cgactcaaga gcgttcgcct
     2641 gagctcactt gataacgtct atatcaaggc cgacaagcag aagaacggca tcaaggcgaa
     2701 cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc actaccagca
     2761 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcgtgca
     2821 gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     2881 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta ccggagggag
     2941 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     3001 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg atgccaccta
     3061 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     3121 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     3181 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac atccaggagc gcaccatctt
     3241 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     3301 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     3361 caagctggag tacaacatgg ttgaccaact gaaagaaaag gacaggaatc tgcgcaggat
     3421 cacccggatg gtgctggtgg tcgtggctgt atttatcgtc tgctggaccc ccatccacat
     3481 ctacgtcatc atcaaagcgc tgatcacgat tccagaaacc acatttcaga ccgtttcctg
     3541 gcacttctgc attgctttgg gttacacgaa cagctgcctg aatccagttc tttacgcctt
     3601 cctggatgaa aacttcaagc gatgcttcag agagttctgc atcccaacct cgtccacgat
     3661 cgaacagcaa aactccactc gagtccgtca gaacactagg gaacatccct ccacggctaa
     3721 tacagtggat cgaactaacc accagctaga aaatctggag gcagaaactg ctccattgcc
     3781 cggcggaggt aagagcagaa tcacaagcga gggcgagtac atccctctgg accagatcga
     3841 catcaacgtg ggagcaccag ccgccgcttt ctgctacgag aacgaagtcg gctaatgtac
     3901 aagtaaagcg gccccggact cgaggccgca ggtaagtatc aaggttacaa gacaggttta
     3961 aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc
     4021 acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcg acaatcaacc
     4081 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     4141 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     4201 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     4261 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     4321 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     4381 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     4441 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     4501 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     4561 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     4621 ccctcagacg agtcggatct ccctttgggc cgcctccccg cctggaattc gagctcggta
     4681 cgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4741 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4801 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4861 aagggggagg attgggaaga caatagccca gcttttgttc cctttagtga gggttaattg
     4921 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctaattc
     4981 actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct
     5041 cacaaatacc actgagatcg atctttttcc ctctgccaaa aattatgggg acatcatgaa
     5101 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     5161 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     5221 tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa
     5281 caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt
     5341 attccataga aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt
     5401 tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct
     5461 ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca
     5521 gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5581 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5641 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5701 cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     5761 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5821 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5881 aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat
     5941 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     6001 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt
     6061 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6121 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6181 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6241 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6301 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6361 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6421 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6481 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6541 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6601 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6661 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6721 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6781 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6841 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6901 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6961 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     7021 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     7081 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     7141 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7201 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7261 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7321 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7381 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7441 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7501 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7561 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7621 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7681 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7741 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7801 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7861 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7921 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7981 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     8041 g
//
