LOCUS       sequence-161464-        5543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting YFP expressed from pJ23117 in the NS1 locus with 
            kanamycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5543)
  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 5543)
  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: pCas27
FEATURES             Location/Qualifiers
     source          1..5543
                     /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=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     misc_RNA        1341..1416
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1437..1466
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1482..1499
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1555..1572)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1725..1771
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1881..1900)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1892..2707
                     /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             1892..2707
                     /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(1965..1984)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(3322..3338)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3322..3338)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3335..3357)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3346..3362
                     /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(3370..3376,3377..3394,3395..3400))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3415..3436
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3553..3570)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3724..4312)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3804..3823)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4483..5274,5275..5343))
                     /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     5106..5125
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5344..5448)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5516..5534
                     /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 tcctagggat tgtgctagct
     1321 ggcatcgccc tcgccctcgc gttttagagc tagaaatagc aagttaaaat aaggctagtc
     1381 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgaagctt gggcccgaac
     1441 aaaaactcat ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt
     1501 gagtttaaac ggtctccagc ttggctgttt tggcggatga gagaagattt tcagcctgat
     1561 acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag
     1621 cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg
     1681 tagtgtgggg tctccccatg cgagagtatg gaactgccag gcatcaaata aaacggaagg
     1741 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac tacggggtct
     1801 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc
     1861 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg
     1921 ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     1981 cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc
     2041 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     2101 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     2161 tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt
     2221 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     2281 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     2341 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     2401 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     2461 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     2521 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     2581 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     2641 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     2701 tttctaagaa ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     2761 taggggttcc gcgcacattt ccccgaaaag gtggatccga aaataaagtc aaagtgcttc
     2821 tataatttaa gatctatttt taaacagtac agctttacag aaagaaaaac cagaagggac
     2881 gcctatttta tctcgttttc ttcccagagt agcaggacat ttttttctgt aaaagcagag
     2941 caatttgtac tgaaatcatt atcaataatg acattagatt cttcgatcaa gacatctgta
     3001 ccgttgctta tatctgcttc agagcctaat acaatggggt attgtggttg ttgacatgca
     3061 gcattaacga gtactttgac tgggaataca accgaatcat cgctataggt gtagacattg
     3121 gcgtggtagt aataggtata gtcatcatag tcgtaattat attcataacc ataccgatac
     3181 tggtaatcaa aatcaataaa tacaggttca ctttttgcct gttgaacaac gataaatgat
     3241 agaccactaa gggctgctgt aaacagttta ttcattttgc tttctccatc ccttgactta
     3301 attaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3361 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3421 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3481 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3541 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3601 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3661 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3721 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3781 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3841 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3901 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3961 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4021 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4081 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4141 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4201 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4261 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4321 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4381 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4441 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4501 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4561 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4621 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4681 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4741 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4801 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4861 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4921 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4981 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5041 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5101 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5161 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5221 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5281 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5341 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5401 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5461 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     5521 gcgtatcacg aggccctttc gtc
//
