LOCUS       sequence-163054-        5541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting YFP (truncated to 18 bp) expressed 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 5541)
  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 5541)
  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: pCas15
FEATURES             Location/Qualifiers
     source          1..5541
                     /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        1339..1414
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1435..1464
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1480..1497
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1553..1570)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1723..1769
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1879..1898)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1890..2705
                     /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             1890..2705
                     /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(1963..1982)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(3320..3336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3320..3336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3333..3355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3344..3360
                     /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(3368..3374,3375..3392,3393..3398))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3413..3434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3551..3568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3722..4310)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3802..3821)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4481..5272,5273..5341))
                     /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     5104..5123
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5342..5446)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5514..5532
                     /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 catcgccctc gccctcgcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     1381 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaagcttgg gcccgaacaa
     1441 aaactcatct cagaagagga tctgaatagc gccgtcgacc atcatcatca tcatcattga
     1501 gtttaaacgg tctccagctt ggctgttttg gcggatgaga gaagattttc agcctgatac
     1561 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg
     1621 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
     1681 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
     1741 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacta cggggtctga
     1801 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg
     1861 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct
     1921 ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     1981 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag
     2041 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     2101 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     2161 ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc caggtattag
     2221 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     2281 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     2341 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     2401 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     2461 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     2521 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     2581 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     2641 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     2701 tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     2761 ggggttccgc gcacatttcc ccgaaaaggt ggatccgaaa ataaagtcaa agtgcttcta
     2821 taatttaaga tctattttta aacagtacag ctttacagaa agaaaaacca gaagggacgc
     2881 ctattttatc tcgttttctt cccagagtag caggacattt ttttctgtaa aagcagagca
     2941 atttgtactg aaatcattat caataatgac attagattct tcgatcaaga catctgtacc
     3001 gttgcttata tctgcttcag agcctaatac aatggggtat tgtggttgtt gacatgcagc
     3061 attaacgagt actttgactg ggaatacaac cgaatcatcg ctataggtgt agacattggc
     3121 gtggtagtaa taggtatagt catcatagtc gtaattatat tcataaccat accgatactg
     3181 gtaatcaaaa tcaataaata caggttcact ttttgcctgt tgaacaacga taaatgatag
     3241 accactaagg gctgctgtaa acagtttatt cattttgctt tctccatccc ttgacttaat
     3301 taagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3361 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3421 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3481 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3541 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3601 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3661 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3721 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3781 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3841 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3901 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3961 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4021 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4081 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4141 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4201 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4261 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4321 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4381 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4441 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4501 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4561 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4621 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4681 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4741 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4801 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4861 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4921 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4981 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5041 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5101 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5161 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5221 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5281 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5341 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5401 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5461 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5521 gtatcacgag gccctttcgt c
//
