LOCUS       sequence-123657-        3070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination plasmid for gRNA concatenation Golden Gate cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3070)
  AUTHORS   Balboa D, Weltner J, Eurola S, Trokovic R, Wartiovaara K, Otonkoski 
            T
  TITLE     Conditionally Stabilized dCas9 Activator for Controlling Gene 
            Expression in Human Cell Reprogramming and Differentiation.
  JOURNAL   Stem Cell Reports. 2015 Sep 8;5(3):448-59. doi: 
            10.1016/j.stemcr.2015.08.001.
  PUBMED    26352799
REFERENCE   2  (bases 1 to 3070)
  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.stemcr.2015.08"; journalName: "Stem Cell Reports"; date: 
            "2015-09-8- 8"; volume: "5"; issue: "3"; pages: "448-59"
COMMENT     Sequence Label: GG-dest
FEATURES             Location/Qualifiers
     source          1..3070
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    166..187
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(202..207,208..225,226..232)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    240..256
                     /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)."
     primer_bind     264..280
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             276..599
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGE
                     FEA"
     primer_bind     299..319
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        301..319
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(367..385)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(395..411)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(646..664)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(683..699)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    707..723
                     /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(731..737,738..755,756..761))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    776..797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(914..931)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1085..1673)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1165..1184)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1844..2635,2636..2704))
                     /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     2467..2486
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2705..2809)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3030..3046
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        join(3054..3070,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3054..3070,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaatacgaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
       61 aagcaagtaa aacctctaca aatgtggtat ggctgattat gatctagagt cgcggccgct
      121 cgttggagac gaccggtgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      181 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      241 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
      301 aattaaccct cactaaaggg aacaaaagct ggaggacctc gagggggggc ccggtaccca
      361 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
      421 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
      481 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      541 atggcgaatg ggacgcgccc tgtagcggcg cccgtctcca gggggaattc gaagcttgag
      601 ctcgagatcc tctagagtcg acctgcaggc atgcaagctt gtctccctat agtgagtcgt
      661 attagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
      721 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
      781 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
      841 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
      901 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      961 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1021 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1081 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1141 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1201 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1261 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1321 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1381 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     1441 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     1501 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     1561 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1621 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1681 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1741 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     1801 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     1861 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     1921 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     1981 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     2041 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     2101 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     2161 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     2221 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     2281 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     2341 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     2401 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     2461 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     2521 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     2581 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     2641 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     2701 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     2761 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     2821 gaaaagtgcc acctgtatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     2881 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     2941 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     3001 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tggatttagg
     3061 tgacactata
//
