LOCUS       sequence-162297-        6428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting YFP +46 from TSS; template strand; 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 6428)
  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 6428)
  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: pCas56
FEATURES             Location/Qualifiers
     source          1..6428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      17..44
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(933..950)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(933..949)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(942..964)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1158..1177)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1277..1299
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1337..1355)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1423..1527
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1528..1596,1597..2388)
                     /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     complement(1746..1765)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2559..3147
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3048..3067
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3301..3318
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3435..3456
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3471..3476,3477..3494,3495..3501)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3509..3525
                     /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)."
     primer_bind     3514..3536
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3533..3549
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3533..3549
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(4164..4979)
                     /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             complement(4164..4979)
                     /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     4887..4906
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4971..4990
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(5100..5146)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5299..5316
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(5372..5389)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5405..5434)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(5455..5530)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5626..5660
                     /label=BBa_J23113
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.01"
     RBS             5669..5680
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5687..6307
                     /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     complement(5760..5779)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             6308..6340
                     /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"
ORIGIN
        1 ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata gttattgata
       61 caaaattaac tcaagaaaaa tagtgtgaac taaaagatac agtaggatat tttaatacag
      121 caatttcatg cctatttgac gaatttctaa gtatagtaat aaaaacactt ggagtcagta
      181 aaaaaacgac tgtgttttag tgctttagaa tgcaaagaca ggcgtcactg taagctgaag
      241 attttttagc cgctcttttt ttattttcat atagcctttg agatattttt cctcactatt
      301 caaaatatta tatttactca gtttttaagt aattagcaga caattctaca tcatcatcta
      361 gcttattgtg agtcgattca ttttgtcggg cttcatttgt gctagaagcc cgtccttgtg
      421 aaaccaaccg aactgcagaa cattgccgcg ccagtactgg ataaacctct ggatctactt
      481 gaatgcctga agaaatcatg tcagcacaaa gtttgactgt ttccgctgaa gattggattt
      541 gaaactttgc taattcggct tgttctcttt ccagtagggt agtgctgaaa tctctacagt
      601 tgctgcccac ttgaccaccg aggggcacaa taatggcacc ggaaagagcg taggaatttg
      661 aactctcccc atcaaatgta ttgccatttg cagagccact gagcacaaag gaagggcggg
      721 gacaaacgat ccctctaaaa ccgtaggtgg cactttgacc taagttgtac acagcggttt
      781 ggttgactac cccagtcgca ctattcccca atgaattttc attattcatc gtattacttt
      841 gatcttgcat cgtttgagcc gctccgtaaa taggagtcag gaacactgtg gagacagccg
      901 ttaaaagtgt gaagaatttt cgaccaaaca tcactggccg tcgttttaca acgtcgtgac
      961 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     1021 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     1081 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     1141 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     1201 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     1261 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     1321 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     1381 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1441 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1501 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1561 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1621 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1681 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1741 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     1801 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1861 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1921 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1981 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2041 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     2101 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2161 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2221 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2281 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2341 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2401 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2461 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2521 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2581 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2641 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2701 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2761 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2821 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2881 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2941 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3001 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3061 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3121 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3181 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3241 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3301 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     3361 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     3421 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     3481 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     3541 agctatgacc atgattacgc caagcttaat taagtcaagg gatggagaaa gcaaaatgaa
     3601 taaactgttt acagcagccc ttagtggtct atcatttatc gttgttcaac aggcaaaaag
     3661 tgaacctgta tttattgatt ttgattacca gtatcggtat ggttatgaat ataattacga
     3721 ctatgatgac tatacctatt actaccacgc caatgtctac acctatagcg atgattcggt
     3781 tgtattccca gtcaaagtac tcgttaatgc tgcatgtcaa caaccacaat accccattgt
     3841 attaggctct gaagcagata taagcaacgg tacagatgtc ttgatcgaag aatctaatgt
     3901 cattattgat aatgatttca gtacaaattg ctctgctttt acagaaaaaa atgtcctgct
     3961 actctgggaa gaaaacgaga taaaataggc gtcccttctg gtttttcttt ctgtaaagct
     4021 gtactgttta aaaatagatc ttaaattata gaagcacttt gactttattt tcggatccac
     4081 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4141 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     4201 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     4261 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     4321 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     4381 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     4441 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     4501 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     4561 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     4621 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     4681 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     4741 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     4801 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     4861 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     4921 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     4981 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     5041 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gttcaccgac aaacaacaga
     5101 taaaacgaaa ggcccagtct ttcgactgag ccttccgttt tatttgatgc ctggcagttc
     5161 catactctcg catggggaga ccccacacta ccatcggcgc tacggcgttt cacttctgag
     5221 ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaatt ctgttttatc
     5281 agaccgcttc tgcgttctga tttaatctgt atcaggctga aaatcttctc tcatccgcca
     5341 aaacagccaa gctggagacc gtttaaactc aatgatgatg atgatgatgg tcgacggcgc
     5401 tattcagatc ctcttctgag atgagttttt gttcgggccc aagcttcaaa aaaagcaccg
     5461 actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt gctatttcta
     5521 gctctaaaac atggtatgtg tctcgtaaat agttaacttt atactctatc actgataggg
     5581 atgtctgtct atcactgata gggatattcc tgcttttttg ttaacctgat ggctagctca
     5641 gtcctaggga ttatgctagc tactagagaa agaggagaaa tactagatgt ccagattaga
     5701 taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt
     5761 aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt
     5821 aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac
     5881 tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag
     5941 ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc
     6001 tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt
     6061 ttcactagag aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt
     6121 attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag
     6181 tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc
     6241 cttcttattc ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag
     6301 tgggtccgct gcaaacgacg aaaactacgc tttagtagct taataacact gatagtgcta
     6361 gtgtagatca ctactagaca aaaaacccct caagacccgt ttagaggccc caaggggttg
     6421 aacgctct
//
