LOCUS       sequence-162311-        6428 bp    DNA     circular SYN 15-APR-2025
DEFINITION  sgRNA targeting YFP +52 from TSS; non-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 Apr 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: pCas52
FEATURES             Location/Qualifiers
     source          1..6428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      152..740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     641..660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     894..911
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1028..1049
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1064..1069,1070..1087,1088..1094)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1102..1118
                     /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     1107..1129
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1126..1142
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1126..1142
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(1757..2572)
                     /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(1757..2572)
                     /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     2480..2499
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2564..2583
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(2693..2739)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2892..2909
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(2965..2982)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2998..3027)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(3048..3123)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             3125..3142
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        3219..3253
                     /label=BBa_J23113
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.01"
     RBS             3262..3273
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3280..3900
                     /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(3353..3372)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             3901..3933
                     /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"
     terminator      4038..4065
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4954..4971)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4954..4970)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4963..4985)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5179..5198)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5298..5320
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5358..5376)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5444..5548
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5549..5617,5618..6409)
                     /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(5767..5786)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
       61 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
      121 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
      181 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
      241 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
      301 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
      361 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      421 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      481 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
      541 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      601 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      661 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      721 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      781 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      841 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      901 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      961 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     1021 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     1081 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     1141 accatgatta cgccaagctt aattaagtca agggatggag aaagcaaaat gaataaactg
     1201 tttacagcag cccttagtgg tctatcattt atcgttgttc aacaggcaaa aagtgaacct
     1261 gtatttattg attttgatta ccagtatcgg tatggttatg aatataatta cgactatgat
     1321 gactatacct attactacca cgccaatgtc tacacctata gcgatgattc ggttgtattc
     1381 ccagtcaaag tactcgttaa tgctgcatgt caacaaccac aataccccat tgtattaggc
     1441 tctgaagcag atataagcaa cggtacagat gtcttgatcg aagaatctaa tgtcattatt
     1501 gataatgatt tcagtacaaa ttgctctgct tttacagaaa aaaatgtcct gctactctgg
     1561 gaagaaaacg agataaaata ggcgtccctt ctggtttttc tttctgtaaa gctgtactgt
     1621 ttaaaaatag atcttaaatt atagaagcac tttgacttta ttttcggatc caccttttcg
     1681 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1741 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     1801 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     1861 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     1921 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     1981 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2041 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2101 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2161 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2221 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2281 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2341 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2401 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2461 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2521 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     2581 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     2641 gagttttcgt tccactgagc gtcagacccc gtagttcacc gacaaacaac agataaaacg
     2701 aaaggcccag tctttcgact gagccttccg ttttatttga tgcctggcag ttccatactc
     2761 tcgcatgggg agaccccaca ctaccatcgg cgctacggcg tttcacttct gagttcggca
     2821 tggggtcagg tgggaccacc gcgctactgc cgccaggcaa attctgtttt atcagaccgc
     2881 ttctgcgttc tgatttaatc tgtatcaggc tgaaaatctt ctctcatccg ccaaaacagc
     2941 caagctggag accgtttaaa ctcaatgatg atgatgatga tggtcgacgg cgctattcag
     3001 atcctcttct gagatgagtt tttgttcggg cccaagcttc aaaaaaagca ccgactcggt
     3061 gccacttttt caagttgata acggactagc cttattttaa cttgctattt ctagctctaa
     3121 aactcatcat catcatcatc atcagttaac tttatactct atcactgata gggatgtctg
     3181 tctatcactg atagggatat tcctgctttt ttgttaacct gatggctagc tcagtcctag
     3241 ggattatgct agctactaga gaaagaggag aaatactaga tgtccagatt agataaaagt
     3301 aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg tttaacaacc
     3361 cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca tgtaaaaaat
     3421 aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca tactcacttt
     3481 tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa aagttttaga
     3541 tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa
     3601 aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg tttttcacta
     3661 gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg cgtattggaa
     3721 gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga tagtatgccg
     3781 ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta
     3841 ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtcc
     3901 gctgcaaacg acgaaaacta cgctttagta gcttaataac actgatagtg ctagtgtaga
     3961 tcactactag acaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttgaacgctc
     4021 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat agttattgat
     4081 acaaaattaa ctcaagaaaa atagtgtgaa ctaaaagata cagtaggata ttttaataca
     4141 gcaatttcat gcctatttga cgaatttcta agtatagtaa taaaaacact tggagtcagt
     4201 aaaaaaacga ctgtgtttta gtgctttaga atgcaaagac aggcgtcact gtaagctgaa
     4261 gattttttag ccgctctttt tttattttca tatagccttt gagatatttt tcctcactat
     4321 tcaaaatatt atatttactc agtttttaag taattagcag acaattctac atcatcatct
     4381 agcttattgt gagtcgattc attttgtcgg gcttcatttg tgctagaagc ccgtccttgt
     4441 gaaaccaacc gaactgcaga acattgccgc gccagtactg gataaacctc tggatctact
     4501 tgaatgcctg aagaaatcat gtcagcacaa agtttgactg tttccgctga agattggatt
     4561 tgaaactttg ctaattcggc ttgttctctt tccagtaggg tagtgctgaa atctctacag
     4621 ttgctgccca cttgaccacc gaggggcaca ataatggcac cggaaagagc gtaggaattt
     4681 gaactctccc catcaaatgt attgccattt gcagagccac tgagcacaaa ggaagggcgg
     4741 ggacaaacga tccctctaaa accgtaggtg gcactttgac ctaagttgta cacagcggtt
     4801 tggttgacta ccccagtcgc actattcccc aatgaatttt cattattcat cgtattactt
     4861 tgatcttgca tcgtttgagc cgctccgtaa ataggagtca ggaacactgt ggagacagcc
     4921 gttaaaagtg tgaagaattt tcgaccaaac atcactggcc gtcgttttac aacgtcgtga
     4981 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     5041 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     5101 tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5161 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5221 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5281 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5341 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5401 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5461 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5521 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5581 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5641 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5701 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5761 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5821 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5881 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5941 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6001 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6061 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6121 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6181 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6241 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6301 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6361 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6421 agtttact
//
