LOCUS       sequence-414012-        3561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 for gRNA in C4 module (5' GCAG-3' TCAG).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3561)
  AUTHORS   Piepers M, Erbstein K, Reyes-Hernandez J, Song C, Tessi T, 
            Petrasiunaite V, Faerber N, Distel K, Maizel A
  TITLE     GreenGate 2.0: Backwards compatible addons for assembly of complex 
            transcriptional units and their stacking with GreenGate.
  JOURNAL   PLoS One. 2023 Sep 8;18(9):e0290097. doi: 
            10.1371/journal.pone.0290097. eCollection 2023.
  PUBMED    37682951
REFERENCE   2  (bases 1 to 3561)
  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: "PLoS One.";
            date: "2023-09-8"; volume: "18(9):e0290097. doi"; pages: " 
            10.1371/journal.pone.0290097. eCollection 2023"
COMMENT     Sequence Label: pKE011
FEATURES             Location/Qualifiers
     source          1..3561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     70..87
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    204..225
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(240..245,246..263,264..270)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    278..294
                     /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        308..326
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     308..325
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        775..850
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1243..1262)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1244..1262)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1272..1294)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1488..1507)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1607..1629
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1667..1685)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1753..1857
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1858..1926,1927..2718)
                     /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     complement(2076..2095)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2889..3477
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3378..3397
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
       61 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
      121 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
      181 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
      241 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
      301 acgagctatt taggtgacac tatagaagtg aagcttggtc tcagcagcgt gtctcaaaat
      361 ctctgatgtt acattgcaca agataaaaat atatcatcat gcctcctcta gaggtgtcgc
      421 tattttactt taaatttttc ttatggctca gcctgtgatg gataactgaa tcaaacaaat
      481 ggcgtctggg tttaagaaca tctgttttgg ctatgttgga cgaaacaagt gaacttttag
      541 gatcaacttc cgtttatata cggagcttat atcgagcaat aagataagtg ggctttttat
      601 gtaatttaat gggctatcgt ccatatattc actaataccc atgcccagta cccatgtatg
      661 cgtttcatat aagctcctaa tttctcccac atcgctcaaa tctaaacaaa tcttgttgta
      721 tatataacac tgagggagca ccattgattg gcaggtggaa ttccacctgc agctgtttta
      781 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      841 gagtcggtgc tttttttgca aaaattttca gattttttct tcatctgtag atttctgggt
      901 ttttttttcc gtttcgtgaa tcataagtga agttttggat gcaaatctgc gcgaaaaaag
      961 ttggacctgc aatgagctta tttagatagc taagacaaag tgattggtcc gttgtttcag
     1021 ttctgattgt cagagagttt gtttcgagtc ggcgacacca atgcgttttg ttaaccagat
     1081 ttcgggtaag aaatgtatcg agagtttgtt tccagacggc tacatcattt tcttatgaag
     1141 ggtgaaatta gatagaccaa agattgaaac acaacatttc tttcacaaaa atataataaa
     1201 cttgatagca tttaggatca gctcagtgag accgaattct cgccctatag tgagtcgtat
     1261 tacctggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     1321 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     1381 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     1441 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     1501 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     1561 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     1621 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     1681 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     1741 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1801 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1861 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1921 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1981 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2041 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     2101 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     2161 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     2221 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     2281 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     2341 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2401 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2461 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2521 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggctctcga
     2581 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2641 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2701 ctgattaagc attggtaact gtcagaccaa gtttactcat atatatttta gattgattta
     2761 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2821 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2881 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2941 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3001 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     3061 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3121 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3181 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3241 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3301 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3361 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3421 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3481 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3541 tttcctgcgt tatcccctga t
//
