LOCUS       sequence-455386-        4570 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Confers puromycin resistance to Salpingoeca rosetta.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4570)
  AUTHORS   Combredet C, Brunet T
  TITLE     A fast and robust gene knockout method for Salpingoeca rosetta 
            clarifies the genetics of choanoflagellate multicellular development
  JOURNAL   bioRxiv 2024.07.13.603360
REFERENCE   2  (bases 1 to 4570)
  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: "bioRxiv 
            2024.07.13.603360"
COMMENT     Sequence Label: pMS18
FEATURES             Location/Qualifiers
     source          1..4570
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(541..546,547..564,565..571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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     584..606
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     603..619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2235..2255
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2573..2590)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2573..2589)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2582..2604)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2798..2817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2917..2939
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2977..2995)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3063..3167
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3168..3236,3237..4028)
                     /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(3386..3405)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4199..4570,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatcttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacaggaaac agctatgacc atgattacgc caagcttgta acgactgcag tcttgttgtc
      661 gtttgtcgtc gtcgttctct gtctcccacc caacttttgc caattgcaac agtgacttct
      721 gtacaatttg caacagatac caacgtgtcg agtcagcaca aatggcgagg aaggttcgcg
      781 tgcattgctt gcgcgcctct ttgtcatggg cgcacgctga tttgtgtgtg atttgcacca
      841 aatttcctgc cgacacaact tgggcccgta ccacacgacc cgtttctgga cccgtttctg
      901 gcccagaaac gcgaatgttg cgaaagatgc gaccacgtgc tcgctgtcga caacacaaac
      961 tcccgcacaa tccccatgaa aaaaaaagtg cttgctgggc ttgtgtctgg cctgtgtgcg
     1021 tggcgtggcc gcgtgtggct tcgttcttgc ttctgctcac tcagcctgtt acacgcagag
     1081 tggctgcgag aaacaaggag aggggtttgc ggcattggcg catgtgcatc tgcctttttc
     1141 attgagtcag tctgcagctc aagaggttgg tttcgtcgac ggatatgagt gagcttctat
     1201 ggatgatgag aggtggtggt ggttgtatgg ttggtggttg gtggttggtg caaggcgagc
     1261 cacgcgaaga gcgacaagga tgagggagag gagaggagac taggaaagag gtgaagggaa
     1321 gggaagggcg agcaatgacg acgagataaa agacagccac caccgcaccc gcacaaaagc
     1381 attgctcgca cgcatggtcg tgtgttatgt ttcacaccac caacataacc aatacaagcc
     1441 atcaccaata caagccagcg ctcaagcaca cacgcacgca cgcacacaca atacacacgc
     1501 acgtgaaacg ttcacattgt ccgctcacaa atgcctttcc cttctcgtcg ctttccttgc
     1561 ctttttttct tcccctcctg acttccttcc tgccttcctt ccttcctccc cttctccgtc
     1621 caccaacacc atttatttct ctgctctttt tcctcgtgcc acgtgagcag tcttttcgcg
     1681 tcgtcttctt ctttgacccc gtttaccgtc aacaccatca acaaccagca acatgtctgg
     1741 tacggagtac aagcctaccg tccgcctcgc tacccgtgac gacgtgcccc gtgctgtccg
     1801 taccctcgcc gctgccttcg ctgactaccc tgctacccgt cacaccgtcg accctgaccg
     1861 ccacatcgag cgtgtcaccg agctgcagga gctgttcctc acccgcgtcg gcctcgacat
     1921 cgggaaggtc tgggtcgccg acgacggtgc tgctgtcgct gtctggacca cccctgagtc
     1981 tgtcgaggcc ggtgctgtct tcgctgagat cggcccccgc atggctgagc tgtctggctc
     2041 ccgtctcgcc gctcagcagc agatggaggg cctcctcgcc ccccaccgcc ccaaggagcc
     2101 cgcctggttc ctcgccaccg tcggcgtctc tcctgaccac cagggcaagg gtctcggctc
     2161 tgccgtcgtc ctccctggcg tcgaggccgc tgagcgtgct ggtgtccctg ctttcctcga
     2221 gacctccgct cctcgcaacc tccccttcta cgagcgtctc ggcttcaccg tcaccgccga
     2281 cgtggaggtc cccgagggtc ctcgtacctg gtgcatgacc cgcaagcccg gtgcttaagc
     2341 gcctcgttgg tcattgccac cttgttgcct cttttgctgt tgtcctcgtg tcgacgcgtg
     2401 ccacgtgcgc ttgttgtgag ttgattctgc ttcttgtgca cgtcatgccg agcccttggg
     2461 ccccgcaata aacccactat gccagtgtaa ctatccttcc ccccaatgta taactgtgac
     2521 gatcaatgag cggtgcccgt ctagaggatc cccgggtacc gagctcgaat tcactggccg
     2581 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     2641 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     2701 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     2761 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     2821 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     2881 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     2941 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     3001 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     3061 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3121 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3181 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3241 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     3301 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3361 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     3421 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     3481 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3541 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3601 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3661 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     3721 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3781 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3841 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3901 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3961 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4021 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4081 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4141 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4201 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4261 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4321 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4381 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4441 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4501 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4561 acaccgaact
//
