LOCUS       sequence-161485-        5550 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting YFP with additional bases 5' end like pCas34, but 
            from pJ23119 in the NS1 locus with kanamycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5550)
  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 5550)
  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: pCas45
FEATURES             Location/Qualifiers
     source          1..5550
                     /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"
     promoter        1285..1319
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        1348..1423
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1444..1473
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1489..1506
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1562..1579)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1732..1778
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1888..1907)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1899..2714
                     /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             1899..2714
                     /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(1972..1991)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(3329..3345)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3329..3345)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3342..3364)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3353..3369
                     /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(3377..3383,3384..3401,3402..3407))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3422..3443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3560..3577)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3731..4319)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3811..3830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4490..5281,5282..5350))
                     /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     5113..5132
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5351..5455)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5523..5541
                     /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 caataacgga attcttgaca gctagctcag tcctaggtat aatgctagct
     1321 gttaactggc atcgccctcg ccctcgcgtt ttagagctag aaatagcaag ttaaaataag
     1381 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttggg
     1441 cccgaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat
     1501 catcattgag tttaaacggt ctccagcttg gctgttttgg cggatgagag aagattttca
     1561 gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg
     1621 gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg
     1681 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa
     1741 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaactac
     1801 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     1861 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     1921 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     1981 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     2041 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     2101 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     2161 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     2221 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     2281 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     2341 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     2401 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     2461 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     2521 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     2581 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     2641 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     2701 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     2761 aaacaaatag gggttccgcg cacatttccc cgaaaaggtg gatccgaaaa taaagtcaaa
     2821 gtgcttctat aatttaagat ctatttttaa acagtacagc tttacagaaa gaaaaaccag
     2881 aagggacgcc tattttatct cgttttcttc ccagagtagc aggacatttt tttctgtaaa
     2941 agcagagcaa tttgtactga aatcattatc aataatgaca ttagattctt cgatcaagac
     3001 atctgtaccg ttgcttatat ctgcttcaga gcctaataca atggggtatt gtggttgttg
     3061 acatgcagca ttaacgagta ctttgactgg gaatacaacc gaatcatcgc tataggtgta
     3121 gacattggcg tggtagtaat aggtatagtc atcatagtcg taattatatt cataaccata
     3181 ccgatactgg taatcaaaat caataaatac aggttcactt tttgcctgtt gaacaacgat
     3241 aaatgataga ccactaaggg ctgctgtaaa cagtttattc attttgcttt ctccatccct
     3301 tgacttaatt aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3361 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3421 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3481 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3541 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3601 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3661 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3721 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3781 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3841 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3901 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3961 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4021 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4081 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4141 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4201 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4261 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4321 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4381 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4441 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4501 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4561 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4621 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4681 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4741 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4801 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4861 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4921 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4981 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5041 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5101 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5161 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5221 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5281 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5341 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5401 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5461 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     5521 aaaataggcg tatcacgagg ccctttcgtc
//
