LOCUS       sequence-162047-        7696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rbcL-sfGFP expressed from PccmK2 promoter with Gmr in glpK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7696)
  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 7696)
  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: pCas49
FEATURES             Location/Qualifiers
     source          1..7696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1034..1052
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1090..1112)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1212..1231
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1425..1447
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1439..1456
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1440..1456
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        join(2241..2246,2247..2263,2264..2269)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             2458..2991
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEVVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     CDS             join(5030..5032,5033..5746)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             5030..5743
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(5060..5088)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5683..5703
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(5798..5816)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5812..5859
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(6536..6552)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6536..6552)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6549..6571)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6560..6576
                     /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(6584..6590,6591..6608,6609..6614))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6629..6650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6767..6784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6938..7526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7018..7037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     1021 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     1081 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     1141 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     1201 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     1261 accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg
     1321 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     1381 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     1441 taaaacgacg gccagtgaat tcgagctcgg tacccgggtg aagcgattgg ctatgatcta
     1501 ccaaagctgg cgatttttct gcaaaccgaa ttagaaccac aatttgattt tttaaagccg
     1561 attaccattg ccaacctgaa ggaaatagcg atcgccttta tggaggatgg ccacgaggct
     1621 tgtcgggtct cccattattg tggatcggta ccggatgagc gggcgagctt taatttacgc
     1681 ttgcggcaat atcgccaggc ccagccttgg ctgcgaaatc atctcgatcc tgcaaggggc
     1741 gatcgcctgc accattggtc ggatcaccaa cgcaccattt tctacggcag gcgcaccaat
     1801 cccgacaccc agcagcggct tgtgttagtg gcgcacatgg ccggggctcc gaagaccgtt
     1861 gagattggca aatggctcgc cctggatttg gatcgttggc agttggcgat cgccacaccg
     1921 actttgaaga tcaacaccat ctatgactta gcccaaattc acttgcacaa tggcgaaggt
     1981 tttctgttat ctgaaattcc tccctaaatg atgtcttttg agcctaaaaa cacacttttt
     2041 tgacctaatt taacccattt aaaaaagctc gagcgcatgc ataaaaactg ttgtaattca
     2101 ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc
     2161 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggacgcaca ccgtggaaac
     2221 ggatgaaggc acgaacccag ttgacataag cctgttcggt tcgtaaactg taatgcaagt
     2281 agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga acgcagcggt ggtaacggcg
     2341 cagtggcggt tttcatggct tgttatgact gtttttttgt acagtctatg cctcgggcat
     2401 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     2461 ttacgcagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaggtggc
     2521 tcaagtatgg gcatcattcg cacatgtagg ctcggccctg accaagtcaa atccatgcgg
     2581 gctgctcttg atcttttcgg tcgtgagttc ggagacgtag ccacctactc ccaacatcag
     2641 ccggactccg attacctcgg gaacttgctc cgtagtaaga cattcatcgc gcttgctgcc
     2701 ttcgaccaag aagtggttgt tggcgctctc gcggcttacg ttctgcccaa gtttgagcag
     2761 ccgcgtagtg agatctatat ctatgatctc gcagtctccg gagagcaccg gaggcagggc
     2821 attgccaccg cgctcatcaa tctcctcaag catgaggcca acgcgcttgg tgcttatgtg
     2881 atctacgtgc aagcagatta cggtgacgat cccgcagtgg ctctctatac aaagttgggc
     2941 atacgggaag aagtgatgca ctttgatatc gacccaagta ccgccaccta acaattcgtt
     3001 caagccgaga tcggcttccc ggccgcggag ttgttcggta aattgtcaca acgccgcagg
     3061 tggcactttt cggggaaatg tgcgcgcccg cgttcctgct ggcgctgggc ctgtttctgg
     3121 cgctggactt cccgctgttc cgtcagcagc ttttcgccca cggccttgat gatcgcggcg
     3181 gccttggcct gcatatcccg attcaacggc cccagggcgt ccagaacggg cttcaggcgc
     3241 tcccgaaggt gaatcctcct ctgacctttg ggcaacttct tctagaaacc cctgtctatt
     3301 taaacgatga agcattgact ctacttaacc ttggtattac gaaatatcat tgcagtttat
     3361 gttgtcaaag gtgccattgt tgcttatgct cttcatagct tgccaaaatc aaaattagta
     3421 gcaaacgcga ttcctggcga tcgccttgtc cactataaag cctgtaaagg accctaaccc
     3481 ctgcgcagag gcaagtggtc ggtcttatgc cgcttccgtt atcccaacta gaagcagacc
     3541 aaactcccag ggggacgcct gttccttagt atttttaaga cattaagctt gataagcgag
     3601 gataaattag atggttcaga ccaaatctgc tgggtttaat gccggtgtac aggactaccg
     3661 cctgacttac tacacccccg attacacccc gaaagatacc gacttactcg cttgtttccg
     3721 gatgactccc caacctggag tcccccccga agaatgtgct gcggctgttg cggctgaatc
     3781 ttctaccggt acttggacca ctgtatggac cgatggttta actgacctcg accgctacaa
     3841 gggtcgttgc tacaatgttg aacccgttcc cggtgaagac aaccaatatt tctgtttcgt
     3901 tgcttacccc ctcgatctgt ttgaagaagg ttctgtaacc aacgttttga cttccttggt
     3961 tggtaacgta ttcggtttta aagcgctgcg tgccctgcgc ctcgaagata tccgcttccc
     4021 cgttgcgtta atcaaaactt accaagggcc tccccacggg atcactgtag agcgtgacct
     4081 cctcaacaag tatggtcgtc ctctcctcgg ttgtacgatt aagccgaagc tcggtctgtc
     4141 tgcgaagaac tacggtcgtg cggtttatga atgtctccgt ggtggtcttg acttcaccaa
     4201 agatgacgaa aacatcaact ctcagccttt catgcgttgg cgcgatcgct tcctgttcgt
     4261 tcaagaagct atcgaaaaat cccaagctga aaccaacgaa gttaagggtc actaccttaa
     4321 cgtcaccgct ggcacttgcg aagaaatgct caagcgggct gaattcgcta aggaaatcgg
     4381 cactcccatc atcatgcacg acttcttaac tggtggtttc actgcgaata ctacccttgc
     4441 gaagtggtgt cgtgataacg gcgttctgct ccacatccac cgggcaatgc acgcggtaat
     4501 cgaccgtcag aagaaccacg gtattcactt ccgcgttctc gctaagtgtc tccgcctctc
     4561 tggtggtgac cacctccact ccggtacggt tgttggtaag ctcgaaggcg atcgcgccgc
     4621 caccctcggt ttcgtagacc tgatgcgtga agactacgtt gaagaagatc gttctcgcgg
     4681 tgtattcttc acccaagact acgcttctct ccccggcacc atgcctgtgg cttccggtgg
     4741 tatccacgta tggcacatgc ctgccctcgt tgaaatcttc ggcgacgatt cctgcctcca
     4801 gtttggtggt ggtaccctcg gtcacccctg gggtaacgca cctggtgcaa ctgcaaaccg
     4861 tgttgctctg gaagcttgtg ttcaagctcg taacgaaggt cgcagcctgg cccgtgaagg
     4921 taatgatgtc ctccgtgaag caggtaagtg gtcgcctgaa ttggcagccg ccctcgacct
     4981 gtggaaggaa atcaagttcg aattcgatac cgttgacact ctcggctcca tgagcaaagg
     5041 agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
     5101 gcacaaattt tctgtccgtg gagagggtga aggtgatgct acaaacggaa aactcaccct
     5161 taaatttatt tgcactactg gaaaactacc tgttccgtgg ccaacacttg tcactactct
     5221 gacctatggt gttcaatgct tttcccgtta tccggatcac atgaaacggc atgacttttt
     5281 caagagtgcc atgcccgaag gttatgtaca ggaacgcact atatctttca aagatgacgg
     5341 gacctacaag acgcgtgctg aagtcaagtt tgaaggtgat acccttgtta atcgtatcga
     5401 gttaaagggt attgatttta aagaagatgg aaacattctt ggacacaaac ttgagtacaa
     5461 ctttaactca cacaatgtat acatcacggc agacaaacaa aagaatggaa tcaaagctaa
     5521 cttcaaaatt cgccacaacg ttgaagatgg ttccgttcaa ctagcagacc attatcaaca
     5581 aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtcgacaca
     5641 atctgtcctt tcgaaagatc ccaacgaaaa gcgtgaccac atggtccttc ttgagtttgt
     5701 aactgctgct gggattacac atggcatgga tgagctctac aaataaggat ccggctgtct
     5761 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     5821 ccccttgggg cctctaaacg ggtcttgacg ggttttttgt ctagatcaac ggcctcaacc
     5881 tactacggat ccccccctct tgcctacagc atctccccca ggggagaatt cttcctgttt
     5941 caactccctc taacgtaaac ccattgaatt taaaaaagac tttatgactg ctttactgct
     6001 ccatgaccaa cattattccc ttgatcatga agcctttctc tcaaccctca gcaacacaga
     6061 aaatttactc attattcaag atctagatgg cgtttgcatg gggttagtca aagacccctt
     6121 aacccgcaaa attgatcctg actatatccg cgccacacgc aagtttagag accacttctt
     6181 tgtcctcacc aacggtgaac atgaaggcag aaggggagta aatcgcatcg ttgaacgggc
     6241 atttcgcaat gttgaagcca aagaggaaac aagctattta cctggtttag cagcaggggg
     6301 tgtgcaatgg cagacagata atggccaaat ttcccatccc ggtgttagcc aagcagaact
     6361 cgatttcctt gccacagtgc cagatttaat tggtcaaagt ttaggacaat tttttactaa
     6421 atatgttgat atttttcccg ctgagcttca acctgagctg atccatgctt ctgttttaga
     6481 taatctcgtt tcctctagag tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat
     6541 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     6601 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     6661 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     6721 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     6781 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6841 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6901 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6961 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     7021 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     7081 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     7141 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     7201 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     7261 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     7321 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     7381 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     7441 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     7501 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     7561 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7621 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7681 aaacttggtc tgacag
//
