LOCUS       sequence-161482-        6427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting cpcB expressed from aTc inducible system in pEZ03 in
            the NS1 locus with kanamycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6427)
  AUTHORS   Gordon GC, Korosh TC, Cameron JC, Markley AL, Begemann MB, Pfleger 
            BF
  TITLE     CRISPR interference as a titratable, trans-acting regulatory tool 
            for metabolic engineering in the cyanobacterium Synechococcus sp. 
            strain PCC 7002.
  JOURNAL   Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 
            10.1016/j.ymben.2016.07.007.
  PUBMED    27481676
REFERENCE   2  (bases 1 to 6427)
  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. 
            2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 
            10.1016/j.ymben.2016.07.007."
COMMENT     Sequence Label: pCas35
FEATURES             Location/Qualifiers
     source          1..6427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(1284..1311)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1416..1448)
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag (LVA)
                     /note="mutant LVA variant that confers accelerated 
                     degradation under some conditions (Andersen et al., 1998)"
                     /translation="AANDENYALVA"
     CDS             complement(1449..2069)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1977..1996
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     RBS             2076..2087
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(2096..2130)
                     /label=BBa_J23113
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.01"
     misc_RNA        2226..2301
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             2322..2351
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2367..2384
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2440..2457)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      complement(2613..2659)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2766..2785)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2777..3592
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2777..3592
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2850..2869)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(4206..4222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4206..4222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4219..4241)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4230..4246
                     /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(4254..4260,4261..4278,4279..4284))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4299..4320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4437..4454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4608..5196)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4688..4707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5367..6158,6159..6227))
                     /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     5990..6009
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6228..6332)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6400..6418
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgatgt ttggtcgaaa attcttcaca
      421 cttttaacgg ctgtctccac agtgttcctg actcctattt acggagcggc tcaaacgatg
      481 caagatcaaa gtaatacgat gaataatgaa aattcattgg ggaatagtgc gactggggta
      541 gtcaaccaaa ccgctgtgta caacttaggt caaagtgcca cctacggttt tagagggatc
      601 gtttgtcccc gcccttcctt tgtgctcagt ggctctgcaa atggcaatac atttgatggg
      661 gagagttcaa attcctacgc tctttccggt gccattattg tgcccctcgg tggtcaagtg
      721 ggcagcaact gtagagattt cagcactacc ctactggaaa gagaacaagc cgaattagca
      781 aagtttcaaa tccaatcttc agcggaaaca gtcaaacttt gtgctgacat gatttcttca
      841 ggcattcaag tagatccaga ggtttatcca gtactggcgc ggcaatgttc tgcagttcgg
      901 ttggtttcac aaggacgggc ttctagcaca aatgaagccc gacaaaatga atcgactcac
      961 aataagctag atgatgatgt agaattgtct gctaattact taaaaactga gtaaatataa
     1021 tattttgaat agtgaggaaa aatatctcaa aggctatatg aaaataaaaa aagagcggct
     1081 aaaaaatctt cagcttacag tgacgcctgt ctttgcattc taaagcacta aaacacagtc
     1141 gtttttttac tgactccaag tgtttttatt actatactta gaaattcgtc aaataggcat
     1201 gaaattgctg tattaaaata tcctactgta tcttttagtt cacactattt ttcttgagtt
     1261 aattttgtat caataactat aaacgcagaa aggcccaccc gaaggtgagc cagtgtgact
     1321 ctagtagaga gcgttcaacc ccttggggcc tctaaacggg tcttgagggg ttttttgtct
     1381 agtagtgatc tacactagca ctatcagtgt tattaagcta ctaaagcgta gttttcgtcg
     1441 tttgcagcgg acccactttc acatttaagt tgtttttcta atccgcatat gatcaattca
     1501 aggccgaata agaaggctgg ctctgcacct tggtgatcaa ataattcgat agcttgtcgt
     1561 aataatggcg gcatactatc agtagtaggt gtttcccttt cttctttagc gacttgatgc
     1621 tcttgatctt ccaatacgca acctaaagta aaatgcccca cagcgctgag tgcatataat
     1681 gcattctcta gtgaaaaacc ttgttggcat aaaaaggcta attgattttc gagagtttca
     1741 tactgttttt ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg atgacttagt
     1801 aaagcacatc taaaactttt agcgttatta cgtaaaaaat cttgccagct ttccccttct
     1861 aaagggcaaa agtgagtatg gtgcctatct aacatctcaa tggctaaggc gtcgagcaaa
     1921 gcccgcttat tttttacatg ccaatacaat gtaggctgct ctacacctag cttctgggcg
     1981 agtttacggg ttgttaaacc ttcgattccg acctcattaa gcagctctaa tgcgctgtta
     2041 atcactttac ttttatctaa tctggacatc tagtatttct cctctttctc tagtagctag
     2101 cataatccct aggactgagc tagccatcag gttaacaaaa aagcaggaat atccctatca
     2161 gtgatagaca gacatcccta tcagtgatag agtataaagt taactgctag aaatgaactc
     2221 acctcgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     2281 aaagtggcac cgagtcggtg ctttttttga agcttgggcc cgaacaaaaa ctcatctcag
     2341 aagaggatct gaatagcgcc gtcgatcatc atcatcatca tcattgagtt taaacggtct
     2401 ccagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga
     2461 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc
     2521 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc
     2581 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggcccag tctttcgact
     2641 gagcctttcg ttttatctgt tgtttgtcgg tgaactacgg ggtctgacgc tcagtggaac
     2701 gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag
     2761 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt
     2821 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     2881 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
     2941 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     3001 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     3061 gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga
     3121 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     3181 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     3241 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     3301 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     3361 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     3421 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     3481 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     3541 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat
     3601 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3661 catttccccg aaaaggtgga tccgaaaata aagtcaaagt gcttctataa tttaagatct
     3721 atttttaaac agtacagctt tacagaaaga aaaaccagaa gggacgccta ttttatctcg
     3781 tttcttccca gagtagcagg acattttttt ctgtaaaagc agagcaattt gtactgaaat
     3841 cattatcaat aatgacatta gattcttcga tcaagacatc tgtaccgttg cttatatctg
     3901 cttcagagcc taatacaatg gggtattgtg gttgttgaca tgcagcatta acgagtactt
     3961 tgactgggaa tacaaccgaa tcatcgctat aggtgtagac attggcgtgg tagtaatagg
     4021 tatagtcatc atagtcgtaa ttatattcat aaccataccg atactggtaa tcaaaatcaa
     4081 taaatacagg ttcacttttt gcctgttgaa caacgataaa tgatagacca ctaagggctg
     4141 ctgtaaacag tttattcatt ttgctttctc catcccttga cttaattaag cttggcgtaa
     4201 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4261 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4321 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4381 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4441 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4501 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4561 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4621 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4681 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4741 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4801 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4861 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4921 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4981 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5041 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5101 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5161 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5221 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5281 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5341 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5401 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5461 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5521 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5581 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5641 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5701 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5761 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5821 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5881 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5941 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6001 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6061 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6121 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6181 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6241 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6301 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6361 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     6421 tttcgtc
//
