LOCUS       sequence-162043-        5185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Delta-ccm:Km, used to delete ccm operon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5185)
  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 5185)
  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: pCas73
FEATURES             Location/Qualifiers
     source          1..5185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     175..197
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(235..253)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        321..425
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(426..494,495..1286)
                     /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(644..663)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1457..2045
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1946..1965
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2199..2216
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2333..2354
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2369..2374,2375..2392,2393..2399)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2407..2423
                     /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     2412..2434
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2431..2447
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2431..2447
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(3329..4144)
                     /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(3329..4144)
                     /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     4052..4071
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4136..4155
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5016..5033)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5016..5032)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5025..5047)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
      121 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
      181 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
      241 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
      301 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
      361 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      421 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      481 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      541 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      601 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      661 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      721 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      781 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      841 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      901 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      961 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     1021 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     1081 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     1141 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     1201 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     1261 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1321 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1381 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1441 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1501 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1561 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1621 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1681 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1741 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1801 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1861 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1921 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1981 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2041 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2101 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2161 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2221 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     2281 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2341 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2401 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2461 agcttgcatg cctgcaggtc gacttattgg ttgtcttcac cgggaacggg ttcaacattg
     2521 tagcaacgac ccttgtagcg gtcgaggtca gttaaaccat cggtccatac agtggtccaa
     2581 gtaccggtag aagattcagc cgcaacagcc gcagcacatt cttcgggggg gactccaggt
     2641 tggggagtca tccggaaaca agcgagtaag tcggtatctt tcggggtgta atcgggggtg
     2701 tagtaagtca ggcggtagtc ctgtacaccg gcattaaacc cagcagattt ggtctgaacc
     2761 atgcggtttt cctccagcaa aaatgcttat ctttaacaga caaataccag taacggtatt
     2821 gttggtcgaa aacttcaaaa atcttacgtt ggcaaaactg ctttttaaaa ttctgaagat
     2881 tcaagtctta tgactttctt taatctgtgg gatatgttac cacagcctcg acttattttt
     2941 atcttttagc aacaaaaaaa gattgctttt gttttttggg ctgattagca tttctaatgc
     3001 tgtttgatgg gcctatttta cccttacaaa aatacctaaa aaagccataa aatcccgctc
     3061 gaattcagtc atgctaccta atgagacatt gggctaaaac cgttttctgt cgggatcttt
     3121 agagagggaa agcgttcaaa tgagatgaat taagtattta tgtttctcaa caatctcctt
     3181 taagaacttt aaacatttaa ttttcactca agcaatcatg aaagttttgt ggggttcagg
     3241 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3301 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     3361 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     3421 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     3481 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     3541 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     3601 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     3661 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     3721 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     3781 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     3841 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     3901 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     3961 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     4021 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     4081 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     4141 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     4201 atcccttaac gtgagttttc gttccactga gcgtcagcta atttatcctc gcttatcaag
     4261 cttaatgtct taaaaatact aaggaacagg cgtccccctg ggagtttggt ctgcttctag
     4321 ttgggataac ggaagcggca taagaccgac cacttgcctc tgcgcagggg ttagggtcct
     4381 ttacaggctt tatagtggac aaggcgatcg ccaggaatcg cgtttgctac taattttgat
     4441 tttggcaagc tatgaagagc ataagcaaca atggcacctt tgacaacata aactgcaatg
     4501 atatttcgta ataccaaggt taagtagagt caatgcttca tcgtttaaat agacaggggt
     4561 ttctagaaga agttgcccaa aggtcagagg actaagcttt tcgctgttga atgaaggcga
     4621 tcgcctgttg gagcgcatct aaacgatttg ttacacgatt gcggggtggc agcaacttta
     4681 acacttctaa tttcctcagg cgatcgcgca cctggcccat ggcccccact aaaaacactt
     4741 cccgtccccg accgtgggca tcccggatca tcgtctccac cgccagggag gccgtcactc
     4801 ccatccgggg tacctcactg aggtctaaaa ttaaaacctc gtatttttca acgatggcca
     4861 tccgttgggc aatccctttc gctgccccaa agctcatggg gccacctaaa caaaaaagaa
     4921 taatgtgtcc ctgggcctgg tgtaacagtt cccgttcgcg ctcatttaac gtatttgtat
     4981 cggccccagg atccccgggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt
     5041 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     5101 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     5161 aatggcgaat ggcgcctgat gcggt
//
