LOCUS       sequence-306381-        3578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the sgRNA targeting to the E. coli can gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3578)
  AUTHORS   Tan SI, Yu PJ, Ng IS
  TITLE     CRISPRi-mediated Programming Essential Gene can as a Direct 
  JOURNAL   Biotechnol Bioeng. 2020 May 27. doi: 10.1002/bit.27443.
  PUBMED    32458463
REFERENCE   2  (bases 1 to 3578)
  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: "Biotechnol 
            Bioeng. 2020 May 27. doi: 10.1002/bit.27443."
COMMENT     Sequence Label: pSUI-SynP-CANTsg
FEATURES             Location/Qualifiers
     source          1..3578
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(669..686)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(840..1428)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(920..939)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1752..2411)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2326..2345
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     2573..2591
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2595..2638)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2641..2662
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     protein_bind    2666..2682
                     /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)."
     misc_RNA        2740..2815
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2822..2842
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     promoter        2889..2966
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(2967..3578,1..471)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2986..3005)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
       61 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
      121 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
      181 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
      241 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
      301 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
      361 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
      421 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      481 aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc
      541 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
      601 gagatctatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
      661 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
      721 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
      781 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      841 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      901 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      961 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1021 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1081 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1141 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc caccggcagc agccactggt
     1201 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1261 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1321 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1381 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1441 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1501 atgagattat caaaaaggat cttcacctag atcctcttaa attaaaaatg aagttttaaa
     1561 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     1621 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     1681 tagataacta cgatacggga gggcttacca tctggctcca gtgctgcaat gataccgcga
     1741 gagtcgacaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
     1801 attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc
     1861 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg
     1921 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
     1981 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
     2041 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
     2101 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
     2161 atcaccagct caccgtcttt cattgccata cgaaattccg gatgagcatt catcaggcgg
     2221 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa
     2281 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat
     2341 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt
     2401 tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc
     2461 ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccc gatcaactcg
     2521 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     2581 tatcacgagg cagaatttca gataaaaaaa atccttagct ttcgctaagg atgatttctg
     2641 gaattcgcgg ccgcttctag aggaattgtg agcggataac aattttgacg gtgtgagcgg
     2701 ataacagtac agtggatcct caggtcagtg tgaatgaccg ttttagagct agaaatagca
     2761 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2821 ttactagtag cggccgctgc agtgcttcct aatgcaggag tcgcataagg gagagcgtcg
     2881 agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     2941 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     3001 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     3061 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     3121 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     3181 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     3241 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     3301 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     3361 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     3421 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     3481 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     3541 tctcggcgcg tctgcgtctg gctggctggc ataaatat
//
