LOCUS       sequence-461752-        5038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA-1 expression from an IPTG-inducible promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5038)
  AUTHORS   Pujar A, Pathania A, Hopper C, Pandi A, Calderon CR, Fugger M, Nowak
            T, Kushwaha M
  TITLE     Phage-mediated intercellular CRISPRi for biocomputation in bacterial
            consortia.
  JOURNAL   Nucleic Acids Res. 2024 Dec 27:gkae1256. doi: 10.1093/nar/gkae1256.
  PUBMED    39727169
REFERENCE   2  (bases 1 to 5038)
  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: "Nucleic 
            Acids Res. 2024 Dec 27:gkae1256. doi: 10.1093/nar/gkae1256."
COMMENT     Sequence Label: pSEVA19_LacI_pLac-sgRNA-1
FEATURES             Location/Qualifiers
     source          1..5038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(14..100)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     115..137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             complement(178..1260)
                     /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     1222..1241
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1261..1338)
                     /gene="lacI"
                     /label=lacI promoter
     protein_bind    1396..1412
                     /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)."
     protein_bind    1419..1435
                     /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        1471..1546
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1611..1705
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        1778..1869
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1870..1938,1939..2730)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      2734..2773
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            2920..3028
                     /note="incP origin of transfer"
     rep_origin      3064..3652
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3553..3572
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3806..3823
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3838..3978
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3904..3923)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4064..4086
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4080..4271)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(4440..4949)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4635..4656)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4843..4862
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tctagggcgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
       61 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctttaa ttaaagcgga
      121 taacaatttc acacaggagg ccgcctaggc cgcggccgcg cgtagaatta tgacaactca
      181 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      241 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
      301 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
      361 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
      421 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
      481 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
      541 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
      601 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
      661 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
      721 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
      781 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
      841 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
      901 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
      961 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1021 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1081 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1141 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     1201 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     1261 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1321 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1381 agcagcccta gaaaattgtg agcggataac aattgacatt gtgagcggat aacaagatac
     1441 tgagcacata atttccgtgc ctgggtatga gttttagagc tagaaatagc aagttaaaat
     1501 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ctgcaggcat
     1561 gcaagcttgc ggccgcgtcg tgactgggaa aaccctggcg actagtcttg gactcctgtt
     1621 gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg
     1681 ccgggcgttt tttattggtg agaatccagg ggtccccaat aattacgatt taaattagta
     1741 gcccgcctaa tgagcgggct tttttttaat tcccctattt gtttattttt ctaaatacat
     1801 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1861 aggaagagta tgagcattca gcattttcgt gtggcgctga ttccgttttt tgcggcgttt
     1921 tgcctgccgg tgtttgcgca tccggaaacc ctggtgaaag tgaaagatgc ggaagatcaa
     1981 ctgggtgcgc gcgtgggcta tattgaactg gatctgaaca gcggcaaaat tctggaatct
     2041 tttcgtccgg aagaacgttt tccgatgatg agcaccttta aagtgctgct gtgcggtgcg
     2101 gttctgagcc gtgtggatgc gggccaggaa caactgggcc gtcgtattca ttatagccag
     2161 aacgatctgg tggaatatag cccggtgacc gaaaaacatc tgaccgatgg catgaccgtg
     2221 cgtgaactgt gcagcgcggc gattaccatg agcgataaca ccgcggcgaa cctgctgctg
     2281 acgaccattg gcggtccgaa agaactgacc gcgtttctgc ataacatggg cgatcatgtg
     2341 acccgtctgg atcgttggga accggaactg aacgaagcga ttccgaacga tgaacgtgat
     2401 accaccatgc cggcagcaat ggcgaccacc ctgcgtaaac tgctgacggg tgagctgctg
     2461 accctggcaa gccgccagca actgattgat tggatggaag cggataaagt ggcgggtccg
     2521 ctgctgcgta gcgcgctgcc ggctggctgg tttattgcgg ataaaagcgg tgcgggcgaa
     2581 cgtggcagcc gtggcattat tgcggcgctg ggcccggatg gtaaaccgag ccgtattgtg
     2641 gtgatttata ccaccggcag ccaggcgacg atggatgaac gtaaccgtca gattgcggaa
     2701 attggcgcga gcctgattaa acattggtaa accgatacaa ttaaaggctc cttttggagc
     2761 cttttttttt ggacgaccct tgtccttttc cgctgcataa ccctgcttcg gggtcattat
     2821 agcgattttt tcggtatatc catccttttt cgcacgatat acaggatttt gccaaagggt
     2881 tcgtgtagac tttccttggt gtatccaacg gcgtcagccg ggcaggatag gtgaagtagg
     2941 cccacccgcg agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg cggtgctcaa
     3001 cgggaatcct gctctgcgag gctggccgta ggccggcccc gtagaaaaga tcaaaggatc
     3061 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3121 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3181 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3241 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3301 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3361 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3421 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3481 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3541 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3601 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3661 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3721 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3781 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3841 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     3901 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3961 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4021 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4081 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4141 tggtcgtgca gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4201 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4261 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4321 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4381 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggggcgcga gattgattta
     4441 ccccggttga taatcagaaa agccccaaaa acaggaagat tgtataagca aatatttaaa
     4501 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4561 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag cccgagatag
     4621 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     4681 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcacccaaat
     4741 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4801 gatttagagc ttgacgggga aagcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     4861 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     4921 cgcgcttaat gcgccgctac agggcgcgta aaaggatcta ggtgaagatc ctttttgata
     4981 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgaggcgcg cccagctg
//
