LOCUS       sequence-162303-        6428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting YFP +172 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 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: pCas55
FEATURES             Location/Qualifiers
     source          1..6428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(415..1206,1207..1275))
                     /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     1038..1057
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1276..1380)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1448..1466
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1504..1526)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1626..1645
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1839..1861
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1853..1870
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1854..1870
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(2759..2786)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2891..2923)
                     /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"
     CDS             complement(2924..3544)
                     /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     3452..3471
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     RBS             3551..3562
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(3571..3605)
                     /label=BBa_J23113
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.01"
     misc_RNA        3701..3776
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             3797..3826
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3842..3859
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3915..3932)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4085..4131
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4241..4260)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4252..5067
                     /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             4252..5067
                     /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(4325..4344)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(5682..5698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5682..5698)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5695..5717)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5706..5722
                     /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(5730..5736,5737..5754,5755..5760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5775..5796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5913..5930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6084..6428,1..244))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6164..6183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
       61 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      121 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      181 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      241 tcaagaagat cctttgatct tttctacggg gtctgatgct cagtggaacg aaaactcacg
      301 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
      361 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
      421 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
      481 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
      541 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
      601 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
      661 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
      721 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
      781 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
      841 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
      901 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
      961 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1021 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1081 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1141 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     1201 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     1261 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     1321 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     1381 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     1441 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     1501 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     1561 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     1621 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     1681 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     1741 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     1801 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     1861 gacggccagt gatgtttggt cgaaaattct tcacactttt aacggctgtc tccacagtgt
     1921 tcctgactcc tatttacgga gcggctcaaa cgatgcaaga tcaaagtaat acgatgaata
     1981 atgaaaattc attggggaat agtgcgactg gggtagtcaa ccaaaccgct gtgtacaact
     2041 taggtcaaag tgccacctac ggttttagag ggatcgtttg tccccgccct tcctttgtgc
     2101 tcagtggctc tgcaaatggc aatacatttg atggggagag ttcaaattcc tacgctcttt
     2161 ccggtgccat tattgtgccc ctcggtggtc aagtgggcag caactgtaga gatttcagca
     2221 ctaccctact ggaaagagaa caagccgaat tagcaaagtt tcaaatccaa tcttcagcgg
     2281 aaacagtcaa actttgtgct gacatgattt cttcaggcat tcaagtagat ccagaggttt
     2341 atccagtact ggcgcggcaa tgttctgcag ttcggttggt ttcacaagga cgggcttcta
     2401 gcacaaatga agcccgacaa aatgaatcga ctcacaataa gctagatgat gatgtagaat
     2461 tgtctgctaa ttacttaaaa actgagtaaa tataatattt tgaatagtga ggaaaaatat
     2521 ctcaaaggct atatgaaaat aaaaaaagag cggctaaaaa atcttcagct tacagtgacg
     2581 cctgtctttg cattctaaag cactaaaaca cagtcgtttt tttactgact ccaagtgttt
     2641 ttattactat acttagaaat tcgtcaaata ggcatgaaat tgctgtatta aaatatccta
     2701 ctgtatcttt tagttcacac tatttttctt gagttaattt tgtatcaata actataaacg
     2761 cagaaaggcc cacccgaagg tgagccagtg tgactctagt agagagcgtt caaccccttg
     2821 gggcctctaa acgggtcttg aggggttttt tgtctagtag tgatctacac tagcactatc
     2881 agtgttatta agctactaaa gcgtagtttt cgtcgtttgc agcggaccca ctttcacatt
     2941 taagttgttt ttctaatccg catatgatca attcaaggcc gaataagaag gctggctctg
     3001 caccttggtg atcaaataat tcgatagctt gtcgtaataa tggcggcata ctatcagtag
     3061 taggtgtttc cctttcttct ttagcgactt gatgctcttg atcttccaat acgcaaccta
     3121 aagtaaaatg ccccacagcg ctgagtgcat ataatgcatt ctctagtgaa aaaccttgtt
     3181 ggcataaaaa ggctaattga ttttcgagag tttcatactg tttttctgta ggccgtgtac
     3241 ctaaatgtac ttttgctcca tcgcgatgac ttagtaaagc acatctaaaa cttttagcgt
     3301 tattacgtaa aaaatcttgc cagctttccc cttctaaagg gcaaaagtga gtatggtgcc
     3361 tatctaacat ctcaatggct aaggcgtcga gcaaagcccg cttatttttt acatgccaat
     3421 acaatgtagg ctgctctaca cctagcttct gggcgagttt acgggttgtt aaaccttcga
     3481 ttccgacctc attaagcagc tctaatgcgc tgttaatcac tttactttta tctaatctgg
     3541 acatctagta tttctcctct ttctctagta gctagcataa tccctaggac tgagctagcc
     3601 atcaggttaa caaaaaagca ggaatatccc tatcagtgat agacagacat ccctatcagt
     3661 gatagagtat aaagttaact cgtcgccgtc cagctcgacc gttttagagc tagaaatagc
     3721 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     3781 tttgaagctt gggcccgaac aaaaactcat ctcagaagag gatctgaata gcgccgtcga
     3841 ccatcatcat catcatcatt gagtttaaac ggtctccagc ttggctgttt tggcggatga
     3901 gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag
     3961 aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
     4021 aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtatg gaactgccag
     4081 gcatcaaata aaacggaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     4141 gtcggtgaac tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4201 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     4261 attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta
     4321 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     4381 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     4441 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     4501 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     4561 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     4621 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     4681 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     4741 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     4801 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     4861 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     4921 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     4981 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     5041 catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg
     5101 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag gtggatccga
     5161 aaataaagtc aaagtgcttc tataatttaa gatctatttt taaacagtac agctttacag
     5221 aaagaaaaac cagaagggac gcctatttta tctcgttttc ttcccagagt agcaggacat
     5281 ttttttctgt aaaagcagag caatttgtac tgaaatcatt atcaataatg acattagatt
     5341 cttcgatcaa gacatctgta ccgttgctta tatctgcttc agagcctaat acaatggggt
     5401 attgtggttg ttgacatgca gcattaacga gtactttgac tgggaataca accgaatcat
     5461 cgctataggt gtagacattg gcgtggtagt aataggtata gtcatcatag tcgtaattat
     5521 attcataacc ataccgatac tggtaatcaa aatcaataaa tacaggttca ctttttgcct
     5581 gttgaacaac gataaatgat agaccactaa gggctgctgt aaacagttta ttcattttgc
     5641 tttctccatc ccttgactta attaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5701 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5761 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5821 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5881 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5941 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6001 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6061 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6121 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6181 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6241 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6301 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6361 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6421 tcgccact
//
