LOCUS       sequence-383089-        5540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses BBa_J23110-sfGFP (capacity monitor) and J3-Bba_J23117-mRFP
            (CRISPRa reporter) on pSC101-AmpR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5540)
  AUTHORS   Kiattisewee C, Karanjia AV, Legut M, Daniloski Z, Koplik SE, Nelson 
            J, Kleinstiver BP, Sanjana NE, Carothers JM, Zalatan JG
  TITLE     Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible
            dCas9 Variants.
  JOURNAL   ACS Synth Biol. 2022 Nov 15. doi: 10.1021/acssynbio.2c00405.
  PUBMED    36378874
REFERENCE   2  (bases 1 to 5540)
  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: "ACS Synth 
            Biol. 2022 Nov 15. doi: 10.1021/acssynbio.2c00405."
COMMENT     Sequence Label: pCK760
FEATURES             Location/Qualifiers
     source          1..5540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(524..1474)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     terminator      complement(2236..2330)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(2360..3151,3152..3220))
                     /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     2983..3002
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3221..3325)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3538..3572
                     /label=BBa_J23110
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.33"
     RBS             3582..3593
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(3600..3602,3603..4316)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             3600..4313
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3630..3658)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4253..4273
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4349..4420
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4436..4463
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4647..4681
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             4691..4702
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4709..5386
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      5419..5490
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5506..5533
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 ctagggtacg ggttttgctg cccgcaaacg ggctgttctg gtgttgctag tttgttatca
       61 gaatcgcaga tccggcttca gccggtttgc cggctgaaag cgctatttct tccagaattg
      121 ccatgatttt ttccccacgg gaggcgtcac tggctcccgt gttgtcggca gctttgattc
      181 gataagcagc atcgcctgtt tcaggctgtc tatgtgtgac tgttgagctg taacaagttg
      241 tctcaggtgt tcaatttcat gttctagttg ctttgtttta ctggtttcac ctgttctatt
      301 aggtgttaca tgctgttcat ctgttacatt gtcgatctgt tcatggtgaa cagctttgaa
      361 tgcaccaaaa actcgtaaaa gctctgatgt atctatcttt tttacaccgt tttcatctgt
      421 gcatatggac agttttccct ttgatatgta acggtgaaca gttgttctac ttttgtttgt
      481 tagtcttgat gcttcactga tagatacaag agccataaga acctcagatc cttccgtatt
      541 tagccagtat gttctctagt gtggttcgtt gtttttgcgt gagccatgag aacgaaccat
      601 tgagatcata cttactttgc atgtcactca aaaattttgc ctcaaaactg gtgagctgaa
      661 tttttgcagt taaagcatcg tgtagtgttt ttcttagtcc gttatgtagg taggaatctg
      721 atgtaatggt tgttggtatt ttgtcaccat tcatttttat ctggttgttc tcaagttcgg
      781 ttacgagatc catttgtcta tctagttcaa cttggaaaat caacgtatca gtcgggcggc
      841 ctcgcttatc aaccaccaat ttcatattgc tgtaagtgtt taaatcttta cttattggtt
      901 tcaaaaccca ttggttaagc cttttaaact catggtagtt attttcaagc attaacatga
      961 acttaaattc atcaaggcta atctctatat ttgccttgtg agttttcttt tgtgttagtt
     1021 cttttaataa ccactcataa atcctcatag agtatttgtt ttcaaaagac ttaacatgtt
     1081 ccagattata ttttatgaat ttttttaact ggaaaagata aggcaatatc tcttcactaa
     1141 aaactaattc taatttttcg cttgagaact tggcatagtt tgtccactgg aaaatctcaa
     1201 agcctttaac caaaggattc ctgatttcca cagttctcgt catcagctct ctggttgctt
     1261 tagctaatac accataagca ttttccctac tgatgttcat catctgagcg tattggttat
     1321 aagtgaacga taccgtccgt tctttccttg tagggttttc aatcgtgggg ttgagtagtg
     1381 ccacacagca taaaattagc ttggtttcat gctccgttaa gtcatagcga ctaatcgcta
     1441 gttcatttgc tttgaaaaca actaattcag acatacatct caattggtct aggtgatttt
     1501 aatcactata ccaattgaga tgggctagtc aatgataatt actagtcctt ttcccgggtg
     1561 atctgggtat ctgtaaattc tgctagacct ttgctggaaa acttgtaaat tctgctagac
     1621 cctctgtaaa ttccgctaga cctttgtgtg ttttttttgt ttatattcaa gtggttataa
     1681 tttatagaat aaagaaagaa taaaaaaaga taaaaagaat agatcccagc cctgtgtata
     1741 actcactact ttagtcagtt ccgcagtatt acaaaaggat gtcgcaaacg ctgtttgctc
     1801 ctctacaaaa cagaccttaa aaccctaaag gcttaagtag caccctcgca agctcgggca
     1861 aatcgctgaa tattcctttt gtctccgacc atcaggcacc tgagtcgctg tctttttcgt
     1921 gacattcagt tcgctgcgct cacggctctg gcagtgaatg ggggtaaatg gcactacagg
     1981 cgccttttat ggattcatgc aaggaaacta cccataatac aagaaaagcc cgtcacgggc
     2041 ttctcagggc gttttatggc gggtctgcta tgtggtgcta tctgactttt tgctgttcag
     2101 cagttcctgc cctctgattt tccagtctga ccacttcgga ttatcccgtg acaggtcatt
     2161 cagactggct aatgcaccca gtaaggcagc ggtatcatca acaggcttac ccgtcttact
     2221 gtccctagtg cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa
     2281 caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct
     2341 cgtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2401 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2461 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     2521 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2581 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2641 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     2701 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     2761 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     2821 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     2881 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     2941 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3001 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     3061 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     3121 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     3181 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     3241 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3301 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gtggcaattc
     3361 cgacgtcagc atttgcgatc attcacgcag cgcttattca gttgctcact gcgatgtcat
     3421 aatcatcgct acgagctgtg aaagatgcat aaagctcgta cgacgcgttc gctcgtctcc
     3481 tcacttctcc tgcggttacc aaaggcgtcc tcgtcgtctt gaagttgcga ttatagattt
     3541 acggctagct cagtcctagg tacaatgcta gcgaattcat taaagaggag aaaggtacca
     3601 tgagcaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
     3661 atgttaatgg gcacaaattt tctgtccgtg gagagggtga aggtgatgct acaaacggaa
     3721 aactcaccct taaatttatt tgcactactg gaaaactacc tgttccgtgg ccaacacttg
     3781 tcactactct gacctatggt gttcaatgct tttcccgtta tccggatcac atgaaacggc
     3841 atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaacgcact atatctttca
     3901 aagatgacgg gacctacaag acgcgtgctg aagtcaagtt tgaaggtgat acccttgtta
     3961 atcgtatcga gttaaagggt attgatttta aagaagatgg aaacattctt ggacacaaac
     4021 tcgagtacaa ctttaactca cacaatgtat acatcacggc agacaaacaa aagaatggaa
     4081 tcaaagctaa cttcaaaatt cgccacaacg ttgaagatgg ttccgttcaa ctagcagacc
     4141 attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc
     4201 tgtcgacaca atctgtcctt tcgaaagatc ccaacgaaaa gcgtgaccac atggtccttc
     4261 ttgagtttgt aactgctgct gggattacac atggcatgga tgagctctac aaataaggat
     4321 ccaaactcga gtaaggatct ccaggcatca aataaaacga aaggctcagt cgaaagactg
     4381 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
     4441 accttcgggt gggcctttct gcgtttataa gtactcagca tttgcgatca ttcacgcagc
     4501 gcttattcag ttgctcactg cgatgtcata atcatcgcta cgagctgtga aagatgcata
     4561 aagctcgtac gacgcgttcg ctcgtctcct cacttctcct acggagcgtt ctggacacaa
     4621 cgtcgtcttg aagttgcgat tatagattga cagctagctc agtcctaggg attgtgctag
     4681 cgaattcatt aaagaggaga aaggtaccat ggcgagtagc gaagacgtta tcaaagagtt
     4741 catgcgtttc aaagttcgta tggaaggttc cgttaacggt cacgagttcg aaatcgaagg
     4801 tgaaggtgaa ggtcgtccgt acgaaggtac ccagaccgct aaactgaaag ttaccaaagg
     4861 tggtccgctg ccgttcgctt gggacatcct gtccccgcag ttccagtacg gttccaaagc
     4921 ttacgttaaa cacccggctg acatcccgga ctacctgaaa ctgtccttcc cggaaggttt
     4981 caaatgggaa cgtgttatga acttcgaaga cggtggtgtt gttaccgtta cccaggactc
     5041 ctccctgcaa gacggtgagt tcatctacaa agttaaactg cgtggtacca acttcccgtc
     5101 cgacggtccg gttatgcaga aaaaaaccat gggttgggaa gcttccaccg aacgtatgta
     5161 cccggaagac ggtgctctga aaggtgaaat caaaatgcgt ctgaaactga aagacggtgg
     5221 tcactacgac gctgaagtta aaaccaccta catggctaaa aaaccggttc agctgccggg
     5281 tgcttacaaa accgacatca aactggacat cacctcccac aacgaagact acaccatcgt
     5341 tgaacagtac gaacgtgctg aaggtcgtca ctccaccggt gcttaaggat ccaaactcga
     5401 gtaaggatct ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     5461 tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt
     5521 gggcctttct gcgtttatac
//
