LOCUS       sequence-368416-        5269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  a pGEM-T easy vector containing the kanamycin resistance cassette 
            flanked by the two regions for the double homologous recombination 
            on the ndhD2 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5269)
  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 5269)
  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-ndhD2.km
FEATURES             Location/Qualifiers
     source          1..5269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     370..389
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(608..712)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(790..1245)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(927..948)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1139..1158
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1371..1393
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1385..1402
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1386..1402
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1409..1428
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1409..1427
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1458..1467
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1462..1471
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2192..2986
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2246..2265)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2856..2875
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        complement(3819..3837)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3820..3837)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3855..3871)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3855..3871)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3868..3890)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3879..3895
                     /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(3903..3909,3910..3927,3928..3933))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3948..3969
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4086..4103)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4257..4845)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4337..4356)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5016..5269,1..538,539..607))
                     /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"
ORIGIN
        1 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
       61 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
      121 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
      181 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
      241 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
      301 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
      361 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
      421 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
      481 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
      541 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
      601 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
      661 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
      721 cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
      781 cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
      841 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
      901 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
      961 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     1021 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     1081 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     1141 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     1201 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
     1261 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     1321 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     1381 acgttgtaaa acgacggcca gtgaattgta atacgactca ctatagggcg aattgggccc
     1441 gacgtcgcat gctcccggcc gccatggcgg ccgcgggaat tcgatttggg acttttagtg
     1501 ctgactttgt tctgtttgtt aaacggattt tggttaccgg cgatcgcctt ctcctggtta
     1561 ttgggttcca gcattttaaa caatggttta attcctttgc tactggaaat tctccagggt
     1621 aatcagaccg gtgtgggcat cggtttatat tttggctgtg tgggtctagc gggggacgtt
     1681 tttacccgtt attggtcaac tcagtccagc cagatgcacg gggggttggg actggcaatg
     1741 ttggtaatgt ctaccttgct ttgtgcccga tattggcaag gattcagagt cccaaacaaa
     1801 aaaggagcag gagattaaat tcccccactc ccctttattt aatttattta agaaaatcac
     1861 acagtagtct caagtagtct gattaactca aaagactaga tcactagggt tgaactttga
     1921 cggtgcccgg gtgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa
     1981 gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg
     2041 gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg
     2101 caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc
     2161 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     2221 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     2281 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     2341 gaccgacctg tccggtgccc tgaatgaact ccaagacgag gcagcgcggc tatcgtggct
     2401 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     2461 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     2521 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     2581 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     2641 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     2701 gttcgccagg ctcaaggcgc ggatgcccga cggcgaggat ctcgtcgtga cccatggcga
     2761 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     2821 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     2881 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     2941 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3001 ttcgctagag gatcgatcct ttttaaccca tcacatatac ctgccgttca ctattattta
     3061 gtgaaatgag atattatgat attttctgaa ttgtgattaa aaaggcaact ttatgcccat
     3121 gcaacagaaa ctataaaaaa tacagagaat gaaaagaaac agatagattt tttagttctt
     3181 taggcccgta gtctgcaaat ccttttatga ttttctatca aacaaaagag gaaaatagac
     3241 cagttgcaat ccaaacgaga gtctaataga atgaggtcga aaagtaaatc gcccgggtaa
     3301 agcaccaaca ttaggaagta gaagaggcgg ggcttatggt ccacctgcca ggcggcaaag
     3361 atggaaaggg tggtaaccag tccggctagt aacactaggg gcatggagat gccatccacg
     3421 gaaacactcc aactgagccc aaattgggga atccagctaa atttttcctg gagctgaaaa
     3481 ccggtactgc tgatgtcgta gttagtccaa aaaacgtagc tcatcaaaac aaaatccgct
     3541 agacccacac caagggcata ccagcggact tgtttcccgt ctttgtcggg aattaacgga
     3601 atgaagaggg cggcgactaa cggcagggca atcatcgttg tcagccaggg gaagtgttcc
     3661 aacatggtaa taagaataaa tacctagtga tcttcctccg attatattaa actttgtaaa
     3721 cttttgagaa ttttttttga gattttccct atagaagtca gagaatggct cgaccatatg
     3781 ggagagctcc caacgcgttg gatgcatagc ttgagtattc tatagtgtca cctaaatagc
     3841 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3901 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3961 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4021 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4081 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4141 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4201 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4261 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4321 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4381 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4441 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4501 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4561 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4621 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4681 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4741 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4801 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4861 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4921 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4981 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5041 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5101 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5161 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5221 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgtt
//
