LOCUS       sequence-316607-        6952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pUC19 backbone with editing template for homologous recombination 
            mediated deletion of Nitrate reductase gene of N. oceanica and 
            selection based on zeocin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6952)
  AUTHORS   Naduthodi MIS, Mohanraju P, Sudfeld C, D'Adamo S, Barbosa MJ, van 
            der Oost J
  TITLE     CRISPR-Cas ribonucleoprotein mediated homology-directed repair for 
            efficient targeted genome editing in microalgae Nannochloropsis 
            oceanica IMET1.
  JOURNAL   Biotechnol Biofuels. 2019 Mar 25;12:66. doi: 
            10.1186/s13068-019-1401-3. eCollection 2019.
  PUBMED    30962821
REFERENCE   2  (bases 1 to 6952)
  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: "Biotechnol 
            Biofuels."; date: "2019-03-25"; volume: "12:66. doi"; pages: " 
            10.1186/s13068-019-1401-3. eCollection 2019"
COMMENT     Sequence Label: pNAD1
FEATURES             Location/Qualifiers
     source          1..6952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1986..2360
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_feature    4284..4340
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4341..4358)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4341..4357)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4350..4372)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4566..4585)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4685..4707
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4745..4763)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4831..4935
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4936..5004,5005..5796)
                     /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(5154..5173)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5967..6555
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6456..6475
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6709..6726
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6843..6864
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6879..6884,6885..6902,6903..6909)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6917..6933
                     /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     6922..6944
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 ctggcgatcg ttgagactaa ccgcggttgt aagccggaac cagctgttgt tcatcactgt
       61 gggtagggaa gggaggggtg gaaaggggag ttcaggggta tgaataaggt gtgacgatta
      121 ctcttactct catagacaca ggtaaagata gagatggatg gaataaaagt atctctagaa
      181 gagaggaaac gaaatactca ctgcctagga cgttccacgt caagtctcga ggctgtgtgt
      241 tctgcgagtt gtcccatgca cgacaggcga cattcgtaca gcccacctat gagagaggga
      301 gggattgtat atatgagagc gtgatggaac acagacgttt ctcgctcgtt ctcaagctgc
      361 actcaccaga cgtaggatgt cgacatcaag attccaaata cgccagcacc agaagcggcc
      421 gtgttcagtt gggtactcct cactgctcag ctgagctagt tcccagctct actcgtggtg
      481 cgtagtcgga atgggtgtgt aaatgtcgat tgagtcaatg atagcagtgt gaaaaccgcg
      541 acaaggtgat acttcctacc ttgccatcgt cgagggagac ctccatgcgt tgcacttgcc
      601 ggccgccacc acagtaggcg tagccttgga gtttgtaagt gcctttcttc agcgggatct
      661 cctcgtcgtg agtctggcag ggaaaatgac gaacagcagt ataagcggcg agcttgcacg
      721 tttgtaaact ttcgtaatcc cagctgaggc ccgcggacac gagtacgtac cggatgggtg
      781 atggccgagt taaggttgag gtcgttgatg atgtactcag gcttgcgcca ccagccctca
      841 ttatccgcgc gctcttggtc caccgacgag ggcaggacgc gattgtcgtg gaagtggtaa
      901 aaggactgcg actcttggcg gctgatagtg atgcgagtaa gccatttgat catccgtccc
      961 cctatgtacc ctgcaagtag aggagataaa aaggaaagtg agtaatatgg ctgtggttca
     1021 tgtatgtatg tgcgtgtatg ggtgtgtatg tgtatgtttg attgtgtacc tggaataatg
     1081 aggcggagtt ggaggggtcg tttatttctc tgccagcctt ggacctcgtg gttgggcgtt
     1141 tcctcctcct tggtgcactg aacctgtccg catcctgcgt cggtttggcg gtcttttgtc
     1201 ctttcctcta tagcccgccc gtctagaggg cacacgcgat gatctttata tctcttcatg
     1261 tgtctttgtt ttaactagga tactgccggg tgaatgccca tcagacaaga ggccaaactc
     1321 tatctacacc cttttgactt ctgttgtggt cgtagtgtgt gcttgcatgc cctgaaagtc
     1381 caggcatccc acttgtgctc taaccccatt caaaacagca gaagtgctta attaagatat
     1441 agattcatga tctcctgtcc cctccttctt accttttcac aaacctcaca cagaagtctc
     1501 cactcttcgc ctctaaaacc tctttttaaa ttatggtaag ttcgtgcggc agtgggtttt
     1561 cggatctata tttgtcaaga tccagttcaa ggtcagggat gtagattaag tacagaagga
     1621 gaagcacaag cgcgccagtt cgcccctcac ggcctggagc agggcattta atccctctat
     1681 cttaccagaa ccatactata caaccaatcc tgttggcatc gctctgtcta tttgtcgtgc
     1741 gtgcatgtgt ccatggtgtg gtggggggca ggggttttcg gggttgcggt tgaaggcacc
     1801 ttatcagaaa gatgccctca gagatagagg tagccccctc cccccgatct tcgaccagtc
     1861 ctgtcaggcg aacactttca cccgtcgttc acctcgttac acacaaggag tagacctctg
     1921 aagttctaat tgtcataaat gcccctcccc cctccctcct tcccttcatc ctcccctccg
     1981 agcagatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     2041 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     2101 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     2161 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     2221 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     2281 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     2341 tggccgagga gcaggactga tggccaggga tcaggaggag ggagtgaaga ggagaaggga
     2401 tctggtttca gagatcccca cttctgccgt cgtctttcgg ccttccttcc ttttaggtgt
     2461 catgccttag gtccttcaag tcctcacctg tcgtcgtcat gtgtgtgtgt gcccgtcata
     2521 caagtcactc gatccaattc acgcatcggt tcaatcaaaa taagactaga ccccgaggga
     2581 agaagggcag aaggaaatcg aaggggtggg atgtgtgtga gagagggaag gagaaatgaa
     2641 agaagtgaac aatgtcatgg tagccagtaa ggagagagta gaagcgaaga aagcaaaagc
     2701 actgttgtga agaaacgaaa tggaagatgg tcatcgctcc tggctctact tgtggttttt
     2761 ctatctttaa tttcaggcgt cctggtctcg ttacatcagc tcccttatct cattggttta
     2821 tcccctactc tactgctgct tcttccttcc atccgtgact gtataacaac gaattgtagt
     2881 accgcagata gacaatagaa aaatgccaaa aaaggcatca ttgatttgct cctccccatt
     2941 aagtcactgt acgccaccat cgccactacc ctcccttagc aaatagtctt tatttagaac
     3001 acagaagaag agtacgttgt gtgcatgcgt gttgttcgta caacttccgt gtcaggacga
     3061 gagaccattt caacagtgag taattccgcc tgcgtttatt ttttcctctt tttccttttc
     3121 ttgttttctt cctttgtacg tgtatttccc tctcttccac gtcttcttta tcgtcatctt
     3181 gtctgccatc cttactctat cttctaccct cactcacttc cacactctca agtccaccct
     3241 ccccctcccc ctcactctcc aacggcgacc agttcgtcca ttgacaaaac ctggggctca
     3301 gctacgccaa ggccggtcac ggttatgcgg tggtcatccc aggcaagctt gggtactggg
     3361 ccgtggttgc tgtaggtgat ggtgtagttt agaaagatga tgagagacac gaactggatt
     3421 ggccgaccca gaaaagaagg acttaccgca catagtgcat ggcggcgggg gtaatgaagc
     3481 cagcgtcgac cagctccttg gtgtgcggct cggcgttaaa cgggtggcga cccgtcaggc
     3541 ggatcatgcc tggcaggcgc tgaacccact tgtctgccgt gtcatcgtct cgcgcgtctg
     3601 tactggtggc acggaccggc tcaggcgccg taggcacctg tggtgagagt ttaaaggcca
     3661 tgtagaggag caacgtgcta aagctgctgc ggcacctgcc ctcgttgtgt ggtgggtggg
     3721 tcatgaagtg tggtcagagt cgggtcagtc atagctgtcc agagtcgacg tatgcagaaa
     3781 cgagaacaca tattgtcgat tcctgctgct acgactgcgc gctaagatcc ggcgccttgc
     3841 agcggttgct tttcctgttc cgacacgtca tgttcgcggc ccccaatcga ggcggcggtt
     3901 ttttggtgca gtgcctgttg tgccaacgcc gtcggtgata aagtaacagg taggtttttg
     3961 gcaaggagtt acacaaacca gtcgaccagg ggacatgaaa tagaacttta cctttcgcaa
     4021 tcgtggatac gtcttgtggg gtacggccga gaagtggcgc aacaaaaaaa gtatgtttcc
     4081 atggtgaccc gcgaaccggg gcagggatgt cgacataaat tgaaaacccg ccgattttca
     4141 accggactta gccaacgtgg tcatctccag ttactcttga gtgggggtat tatgatttgt
     4201 cttgcttgtt tgcttcttta tgaaagcttg gcattgtttt ttccaaagca cctaatgaca
     4261 tgccgcatga ccatgattac gccaagcttg catgcctgca ggtcgactct agaggatccc
     4321 cgggtaccga gctcgaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4381 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4441 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     4501 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     4561 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4621 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4681 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4741 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4801 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4861 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4921 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4981 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5041 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5101 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5161 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5221 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5281 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5341 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5401 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5461 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5521 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5581 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5641 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5701 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5761 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5821 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5881 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5941 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6001 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6061 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6121 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6181 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6241 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6301 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6361 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6421 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6481 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6541 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6601 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6661 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     6721 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     6781 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     6841 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     6901 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ct
//
