LOCUS       sequence-371418-        5194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG inducible GFP with sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5194)
  AUTHORS   Kim SK, Kim H, Woo SG, Kim TH, Rha E, Kwon KK, Lee H, Lee SG, Lee DH
  TITLE     CRISPRi-based programmable logic inverter cascade for 
            antibiotic-free selection and maintenance of multiple plasmids.
  JOURNAL   Nucleic Acids Res. 2022 Dec 9;50(22):13155-13171. doi: 
            10.1093/nar/gkac1104.
  PUBMED    36511859
REFERENCE   2  (bases 1 to 5194)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2022-12-9- 9"; volume: "50"; issue: 
            "22"; pages: "13155-13171"
COMMENT     Sequence Label: pT-GFP-rG2
FEATURES             Location/Qualifiers
     source          1..5194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(193..198,199..215,216..222)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    230..246
                     /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     235..257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             910..957
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             958..981
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     terminator      1019..1090
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1106..1133
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        1183..1217
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1238..1313
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1378..1395)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1548..1634
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1726..1753
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1772..1863
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1864..1932,1933..2724)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2082..2101)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2895..3483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3384..3403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3637..3654
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3669..3809
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3735..3754)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3895..3917
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3995..4072
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4073..5155
                     /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(4092..4111)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
       61 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
      121 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
      181 tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga
      241 taacaatttc acacaggagg ttaaaccatg agcaaaggtg aagaactgtt taccggcgtt
      301 gtgccgattc tggtggaact ggatggcgat gtgaacggtc acaaattcag cgtgcgtggt
      361 gaaggtgaag gcgatgccac gattggcaaa ctgacgctga aatttatctg caccaccggc
      421 aaactgccgg tgccgtggcc gacgctggtg accaccctga cctatggcgt tcagtgtttt
      481 agtcgctatc cggatcacat gaaacgtcac gatttcttta aatctgcaat gccggaaggc
      541 tatgtgcagg aacgtacgat tagctttaaa gatgatggca aatataaaac gcgcgccgtt
      601 gtgaaatttg aaggcgatac cctggtgaac cgcattgaac tgaaaggcac ggattttaaa
      661 gaagatggca atatcctggg ccataaactg gaatacaact ttaatagcca taatgtttat
      721 attacggcgg ataaacagaa aaatggcatc aaagcgaatt ttaccgttcg ccataacgtt
      781 gaagatggca gtgtgcagct ggcagatcat tatcagcaga ataccccgat tggtgatggt
      841 ccggtgctgc tgccggataa tcattatctg agcacgcaga ccgttctgtc taaagatccg
      901 aacgaaaaac gggaccacat ggttctgcac gaatatgtga atgcggcagg tattacgtgg
      961 agccatccgc agttcgaaaa ataatctaga gtcgacctgc ataaggatct ccaggcatca
     1021 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     1081 gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct gcgtttatac
     1141 ctagggatat attccgcttc ctcggcgacc ggttaaagat ctttgacagc tagctcagtc
     1201 ctaggtataa tactagtaga cgctgattac atggactgtt ttagagctag aaatagcaag
     1261 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     1321 gacccgggtg caggcatgca agcttggctg ttttggcgga tgagagaaga ttttcagcct
     1381 gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag
     1441 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
     1501 tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
     1561 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     1621 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     1681 ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga
     1741 cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat
     1801 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     1861 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     1921 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     1981 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2041 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2101 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2161 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2221 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2281 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2341 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2401 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2461 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     2521 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     2581 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     2641 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     2701 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     2761 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     2821 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2881 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2941 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3001 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3061 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3121 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3181 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3241 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3301 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3361 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3421 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3481 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3541 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3601 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3661 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3721 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3781 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3841 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3901 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt
     3961 cgcgcgcgaa ggcgaagcgg catgcattta cgttgacacc atcgaatggt gcaaaacctt
     4021 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     4081 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     4141 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4201 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     4261 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     4321 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     4381 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     4441 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     4501 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     4561 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     4621 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     4681 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     4741 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     4801 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     4861 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     4921 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     4981 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     5041 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     5101 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     5161 acgcaattaa tgtaagttag cgcgaattga tctg
//
