LOCUS       sequence-163053-        5535 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting YFP (truncated to 12 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 5535)
  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 5535)
  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: pCas13
FEATURES             Location/Qualifiers
     source          1..5535
                     /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        1333..1408
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1429..1458
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1474..1491
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1547..1564)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1717..1763
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1873..1892)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1884..2699
                     /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             1884..2699
                     /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(1957..1976)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(3314..3330)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3314..3330)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3327..3349)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3338..3354
                     /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(3362..3368,3369..3386,3387..3392))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3407..3428
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3545..3562)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3716..4304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3796..3815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4475..5266,5267..5335))
                     /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     5098..5117
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5336..5440)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5508..5526
                     /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 ggcatcgccc tcgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     1381 acttgaaaaa gtggcaccga gtcggtgctt tttttgaagc ttgggcccga acaaaaactc
     1441 atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttaa
     1501 acggtctcca gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta
     1561 aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
     1621 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg
     1681 ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     1741 aaagactggg cctttcgttt tatctgttgt ttgtcggtga actacggggt ctgacgctca
     1801 gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca
     1861 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc
     1921 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     1981 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
     2041 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     2101 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     2161 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat
     2221 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     2281 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     2341 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     2401 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     2461 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     2521 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     2581 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     2641 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag
     2701 aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2761 ccgcgcacat ttccccgaaa aggtggatcc gaaaataaag tcaaagtgct tctataattt
     2821 aagatctatt tttaaacagt acagctttac agaaagaaaa accagaaggg acgcctattt
     2881 tatctcgttt tcttcccaga gtagcaggac atttttttct gtaaaagcag agcaatttgt
     2941 actgaaatca ttatcaataa tgacattaga ttcttcgatc aagacatctg taccgttgct
     3001 tatatctgct tcagagccta atacaatggg gtattgtggt tgttgacatg cagcattaac
     3061 gagtactttg actgggaata caaccgaatc atcgctatag gtgtagacat tggcgtggta
     3121 gtaataggta tagtcatcat agtcgtaatt atattcataa ccataccgat actggtaatc
     3181 aaaatcaata aatacaggtt cactttttgc ctgttgaaca acgataaatg atagaccact
     3241 aagggctgct gtaaacagtt tattcatttt gctttctcca tcccttgact taattaagct
     3301 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3361 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     3421 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3481 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3541 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3601 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3661 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3721 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3781 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3841 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3901 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3961 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4021 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4081 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4141 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4201 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4261 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4321 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4381 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4441 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4501 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4561 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4621 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4681 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4741 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4801 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4861 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4921 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4981 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5041 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5101 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5161 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5221 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5281 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5341 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5401 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5461 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     5521 cgaggccctt tcgtc
//
