LOCUS       sequence-137247-        2705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for expression of gRNA in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2705)
  AUTHORS   Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang YJ, Chen T, Zhao X
  TITLE     Metabolic engineering of Escherichia coli using CRISPR-Cas9 
            meditated genome editing.
  JOURNAL   Metab Eng. 2015 Sep;31:13-21. doi: 10.1016/j.ymben.2015.06.006. Epub
            2015 Jun 30.
  PUBMED    26141150
REFERENCE   2  (bases 1 to 2705)
  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: "Metab 
            Eng."; date: "2015-09"; volume: "31:13-21. doi"; pages: " 
            10.1016/j.ymben.2015.06.006. Epub 2015 Jun 30"
COMMENT     Sequence Label: pGRB
FEATURES             Location/Qualifiers
     source          1..2705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..35
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(59..81)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(176..493)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDA
                     A"
     primer_bind     181..200
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     394..416
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     408..425
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     409..425
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    429..485
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(494..510)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(494..510)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(507..529)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    518..534
                     /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(542..548,549..566,567..572))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    587..608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(724..741)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(895..1483)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(975..994)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1654..2445,2446..2514))
                     /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     2277..2296
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2515..2619)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2687..2705
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttgacagcta gctcagtcct aggtataata ctagtttttt cggtgatgac ggtgaaaacc
       61 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
      121 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
      181 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
      241 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg
      301 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
      361 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
      421 ccagtgccaa gcttgcatgc ctgcaggtcg actctagagg atccccgggt accgagctcg
      481 aattcgtaat catgtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
      541 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
      601 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcagttttag
      661 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccc
      721 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
      781 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
      841 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      901 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      961 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1021 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1081 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1141 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1201 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1261 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1321 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1381 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1441 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1501 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1561 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1621 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1681 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1741 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgtgaccc
     1801 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1861 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1921 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1981 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2041 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2101 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2161 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2221 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     2281 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     2341 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     2401 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2461 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2521 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     2581 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     2641 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     2701 gaggc
//
