LOCUS       sequence-135053-        6094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains sgRNA which targets GFPmut3b. Under an aTc inducible 
            promoter. Suicide vector inserts into slr0230 site of Synechocystis.
            Carries kanamycin resistance. Propogates in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6094)
  AUTHORS   Yao L, Cengic I, Anfelt J, Hudson EP
  TITLE     Multiple Gene Repression in Cyanobacteria Using CRISPRi.
  JOURNAL   ACS Synth Biol. 2015 Dec 28.
  PUBMED    26689101
REFERENCE   2  (bases 1 to 6094)
  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: "ACS Synth 
            Biol. 2015 Dec 28."
COMMENT     Sequence Label: pMD19T-slr0230-PL22-sgRNANT1-KmR
FEATURES             Location/Qualifiers
     source          1..6094
                     /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"
     misc_feature    1476..1497
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     protein_bind    1498..1516
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1523..1541
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        1575..1650
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1674..1745
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1761..1788
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1839..2654)
                     /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(1839..2654)
                     /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     2562..2581
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2646..2665
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     misc_feature    2769..2789
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(3873..3889)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3873..3889)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3886..3908)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3897..3913
                     /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(3921..3927,3928..3945,3946..3951))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3966..3987
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4104..4121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4275..4863)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4355..4374)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5034..5825,5826..5894))
                     /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     5657..5676
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5895..5999)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6067..6085
                     /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 ccagtgaatt agaactcggt acgcgcggat
      421 cttccagaga tttgccacct gacgtctaag aaaccataac cgttgtccct tttgtttcat
      481 cgaccaacaa ccgccgggga aacgggagag tttgtattac aaagatgatg actatcgcct
      541 gagttttctc tacggcagtt atctcacttt gactaatttg agtgccaagg aatggcaacg
      601 catagaacag ttaaggcttt ctcccctcta cgtttccatc catgccacgg aagcatcggt
      661 gcgggaaaga ttgttaaaaa atcatcgggc cggtcagatt cttgaccagt tggcttggtt
      721 ccaagctaga agattacaga tccacgccca ggtggtggtt tgtcctggtg ttaatgatgg
      781 taagcactta gagcaaactt tgagggactt agcccagttt caccaagggg aaaccccagc
      841 ggtgatttca gtggcggtgg tgccggtggg tctaacccgt tttcgtcccc aggaagatga
      901 gttaagcccc gttggtcaag ctaaagccac ggaggtaatt gcccaggttc aagctttgca
      961 aaaggaattt gcccagcaac taggtggtaa ctttgtttgg ttggccgatg aatggtttct
     1021 cattgcccgt caacctttgc caccggaaag ccattacgaa gactatcccc aaattggcaa
     1081 cggggtaggg tctatccgtc aattcattaa ggagtttcaa caacaagctg cggaatttct
     1141 ccccccggcg atcgccgaag ccaagacgtt gacctgggta gtgggcaatg ctgtagaaca
     1201 agcgtttgag ctactggtgg aacagttaaa tcaggtaaag ggtttaacag ttaatttagc
     1261 gcccttaaac agtgactatt ggggtcagga aattacggtg acgggactat tgacggggca
     1321 ggatttaatc gctaaattgg caggcagaga tttaggggat ggtattctat tgcctgctct
     1381 gatgttgaaa catgatgata ctcgcttctt ggatgacctc cgggtggccg atgtggctca
     1441 aaagttgggc acaaccattt accctgtggc tgatggaatt cgcggccgct tctagagtcc
     1501 ctatcagtga tagagattga catccctatc agtgatagag atactgggag ctaccatcta
     1561 attcaacaag aattgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     1621 caacttgaaa aagtggcacc gagtcggtgc ttttttttac tagagccagg catcaaataa
     1681 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     1741 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatatactag
     1801 aggctctgcc agtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc
     1861 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     1921 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     1981 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     2041 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     2101 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     2161 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     2221 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     2281 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     2341 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     2401 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     2461 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     2521 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     2581 ccatataaat cagcatccat gttggaattt aatcgcggcc tggagcaaga cgtttcccgt
     2641 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     2701 catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg
     2761 ctttgttgta ctagtagcgg ccgctgcagc ggagtttaca taatctgaat cagtttctgg
     2821 aattgacccc tctgatggcc accagggtca ccgatgccga tggcagtgct ttagttctaa
     2881 tgagagaggg ggaaatatct atctttgaac aaatccatgg ccacaaaaat agtcttaagg
     2941 gcaccattaa aggcgctttg cagaaggccc gccaggttaa cctcacattg gattcttcca
     3001 ctgtcctgag ttattttgac cgtcaattgc gccaagaatt accggcgatc gcctgttata
     3061 acacccccat tttgagtcac caagaggaag tgggccgctt atatatcttt agtcaagatc
     3121 gtaactatag ctggactccc acccgtcgta agttactaca gctaatttct gatcaaaccg
     3181 ctgtggcgat cgccaatagt gatttaaacc aaaagttgag atccagagaa agtcaggatc
     3241 gggaattgga aattgcttca gaaattcaaa atcaactatt gccccgttgt tgtccccaaa
     3301 ttaatggttt ggacattgct gcccagtgta aaaccgccag tcgggtaggt ggggattact
     3361 acgatttcat tcctgccaac tatgaccaac tgcgccaggg ggattggtta tgtcgtaaca
     3421 caagccattt gggggtgccc tggagtattg tcatcggcga tgtcatgggt aaaggagtac
     3481 cggcgggtct aattatgacc atgactaggg ggatgctgcg ggcagaagtg ctgaaccgcc
     3541 atagtccagc ccaaattctt aaccacctca accgggtaat gtatgccgac ctggaaaatt
     3601 cccatcgctt tgtcaccttg ttttattctg aatataaccc cgaaacgagc atactttcct
     3661 atagcaacgc cgcccataac cctcccctat tgtggcgggc cggcagtgac agcccccaat
     3721 gtttaattcc cctggatacg gaaggagctt taatcggctt ggagtcggat tccaattacc
     3781 gggatgccca gattcagctt gttcccccag tcgggaaacc tgtcgtgcca gaaatcgtcg
     3841 aacggcaggc gtgcaaactt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3901 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3961 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4021 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4081 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4141 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4201 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4261 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4321 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4381 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4441 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4501 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4561 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4621 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4681 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4741 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4801 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4861 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4921 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4981 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5041 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5101 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5161 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5221 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5281 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5341 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5401 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5461 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5521 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5581 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5641 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5701 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5761 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5821 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5881 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5941 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatggg ggttccgcgc
     6001 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     6061 tataaaaata ggcgtatcac gaggcccttt cgtc
//
