LOCUS       sequence-371409-        5543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  a pGEM-T easy vector containing the streptomycin/spectinomycin 
            resistance cassette flanked by the two regions for the double 
            homologous recombination on the flv3 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5543)
  AUTHORS   Spasic J, Oliveira P, Pacheco C, Kourist R, Tamagnini P
  TITLE     Engineering cyanobacterial chassis for improved electron supply 
            toward a heterologous ene-reductase.
  JOURNAL   J Biotechnol. 2022 Dec 10;360:152-159. doi: 
            10.1016/j.jbiotec.2022.11.005. Epub 2022 Nov 9.
  PUBMED    36370921
REFERENCE   2  (bases 1 to 5543)
  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: "J 
            Biotechnol."; date: "2022-12-10"; volume: "360:152-159. doi"; pages:
            " 10.1016/j.jbiotec.2022.11.005. Epub 2022 Nov 9"
COMMENT     Sequence Label: pGEM_Delta-flv3.sm
FEATURES             Location/Qualifiers
     source          1..5543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2..24)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13..29
                     /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(37..43,44..61,62..67))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    82..103
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(220..237)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(391..979)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(471..490)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1150..1941,1942..2010))
                     /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     1773..1792
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2011..2115)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2193..2648)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2330..2351)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2542..2561
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2774..2796
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2788..2805
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2789..2805
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2812..2831
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2812..2830
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2861..2870
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2865..2874
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     terminator      3636..3730
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3966..4757
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(5496..5514)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5497..5514)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(join(5532..5543,1..5))
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(join(5532..5543,1..5))
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
       61 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
      121 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
      181 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
      241 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
      301 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
      361 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
      421 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
      481 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
      541 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
      601 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
      661 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      721 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
      781 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
      841 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
      901 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
      961 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     1021 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     1081 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1141 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1201 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1261 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     1321 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1381 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1441 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1501 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     1561 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     1621 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     1681 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     1741 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     1801 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     1861 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1921 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1981 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     2041 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2101 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc
     2161 acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa
     2221 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2281 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     2341 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2401 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     2461 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     2521 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2581 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     2641 gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     2701 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     2761 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac
     2821 tcactatagg gcgaattggg cccgacgtcg catgctcccg gccgccatgg cggccgcggg
     2881 aattcgattc tcaccgaaca ttgccattat cctagtctcc ggccccaact ttggactggc
     2941 attgaaatta ccgccaattt agacggcact gaagtgcaca tcctgggcta tgactttgac
     3001 cctacccacc cggtcctaga cccctatcta accggcgatc gcccggataa gtactatgcc
     3061 caagcgccca aagtcattaa ggctctccat gatgccggtg gcttagtggt gttggcccat
     3121 ccagcccggt accgtcagcc cgccagtcac ttgatcccag cggcagcccg ccatggcatt
     3181 gatggggtag aggcatttta tgcctatggc aatcccaaac cttggcagag ttctgtgccc
     3241 caaatggaag aagtttgtta cctgggggat ttgtataatc tgttccgcac ctgtggcacc
     3301 gatagccatg gggaaagcat catgtatcgt ctctgaagtc tctagggaaa gtcgtaatcg
     3361 ttagagttaa aaatatcaat gtttgttaac tttccccggc tctggcccct gggatacact
     3421 ggacagagag ttttcattac cagtcctagg agtacggcat gttcactacc cccctccccc
     3481 cccaaaagcg gctttctacc caaactgagg cgatcgccaa aaatattact gctatccgtt
     3541 ctttggactg ggcccgggga tcctctagag tcgacctgca ggcatgcaag cttgcggccg
     3601 cgtcgtgact gggaaaaccc tggcgactag tcttggactc ctgttgatag atccagtaat
     3661 gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat
     3721 tggtgagaat ccaggggtcc ccaataatta cgatttacgt atttaaatga accttgaccg
     3781 aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg
     3841 gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt
     3901 tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttaa
     3961 acatcatgag ggaagcggtg atcgccgaag tatcgactca actatcagag gtagttggcg
     4021 tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg
     4081 atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc gtaaggcttg
     4141 atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct tcccctggag
     4201 agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac atcattccgt
     4261 ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat gacattcttg
     4321 caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg acaaaagcaa
     4381 gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat ccggttcctg
     4441 aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg ccgcccgact
     4501 gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac agcgcagtaa
     4561 ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc ctgccggccc
     4621 agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa gaagatcgct
     4681 tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc gagatcacca
     4741 aggtagtcgg caaataagac aagggatcgc tttgtgttga acattttgga ggaaggcaac
     4801 tatcaaattt tgatgaaaca tttcctcaaa cgtttccccc ctggtgctga tcgttttgct
     4861 ggagtaaaaa cccaaaccgc aagcaacgga tcgcctattt tgaccgatgc gctagcctat
     4921 ttggagtgtg aagtagctag tcggatggaa tgtagtgatc actggattgt ttacagccaa
     4981 gtaaccaatg gtcgggtagc taaagcagaa gggttaaccg ccgtacacca tcgcaaagtc
     5041 ggcaattatt actaataaaa aactgcaatc aaaccgcaaa actatcaagg aaaatccccc
     5101 aggccatctc tagcttgagg ggattttgtt cggggaatga tggcgtcgct ataccgccag
     5161 acgatggatc actttgttag tcggcttaat ccttgggtag gtctaactaa tttctcaggc
     5221 catatttaaa atccatattg ccttcatagc cagtgagctc tgccaattcc cggagagttt
     5281 tgaccccagg gtttagttct ccgtcaatgc cccaggtggg gaaggattga ataccgacat
     5341 ccacacaggt ttgggtctgg ggatttttcc cggccgggtc gcactcaata tagctaattt
     5401 tttcaaatgc ttctttgccg aaaagtagtt tttggtcata gcagtctcga ccatatggga
     5461 gagctcccaa cgcgttggat gcatagcttg agtattctat agtgtcacct aaatagcttg
     5521 gcgtaatcat ggtcatagct gtt
//
