LOCUS       sequence-489760-        4661 bp    DNA     circular SYN 20-OCT-2025
DEFINITION  In-Vitro Synthesis of mRNA encoding a Honeybee-optimized 
            mTagBFP2-labeled Nuclear Localization Signal (NLS) Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4661)
  TITLE     In-Vitro Synthesis of mRNA for Fluorescent Labeling of Honeybee 
            Embryos
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4661)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 20, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pBSII-NLS-mTagBFP(AM)
FEATURES             Location/Qualifiers
     source          1..4661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             729..749
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1584..1718
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1621..1640)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1675..1694
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2473..2493)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2473..2491)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2512..2528)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2512..2528)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2525..2547)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2536..2552
                     /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(2560..2566,2567..2584,2585..2590))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2605..2626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2743..2760)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2914..3502)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2994..3013)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3673..4464,4465..4533))
                     /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     4296..4315
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4534..4638)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccgggca
      721 aaatggcccc caagaagaaa aggaaagtgg cggccgccat gtcgaaaggt gaagaattga
      781 ttaaagaaaa catgcatatg aaattgtaca tggaaggcac cgtggacaac caccacttca
      841 aatgcacctc ggaaggcgaa ggcaaaccat acgaaggcac ccaaacaatg agaattaaag
      901 tggtggaagg tggcccattg ccattcgcct tcgacatttt ggccacctcg ttcttgtacg
      961 gatcgaaaac cttcattaac cacacccaag gcattccaga tttcttcaaa caatcgttcc
     1021 cagaaggctt cacctgggaa agagtgacaa cctacgaaga cggaggagtg ttgaccgcca
     1081 cccaagacac ctcgttgcaa gacggatgct tgatttacaa cgtgaaaatt agaggagtga
     1141 acttcacatc gaatggccca gtgatgcaaa agaaaacctt gggttgggaa gccttcaccg
     1201 aaaccttgta cccagccgat ggtggtttgg aaggcagaaa cgacatggcg ttgaaattgg
     1261 tgggcggctc gcacttgatt gccaacgcca aaaccaccta cagatcgaaa aaaccggcca
     1321 aaaacttgaa aatgccaggt gtgtactacg tggactacag attggaaaga attaaagaag
     1381 ccaacaacga aacctacgtg gaacaacatg aagtggcggt ggcgagatac tgcgacttgc
     1441 cgtcgaaatt gggccacaaa ttgaactaac ctgcagggat cataatcagc cataccacat
     1501 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     1561 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     1621 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     1681 tgtccaaact catcaatgta tcttatcatg tctggatccc caggaagctc ctctgtgtcc
     1741 tcataaaccc taacctcctc tacttgagag gacattccaa tcataggctg cccatccacc
     1801 ctctgtgtcc tcctgttaat taggtcactt aacaaaaagg aaattgggta ggggtttttc
     1861 acagaccgct ttctaagggt aattttaaaa tatctgggaa gtcccttcca ctgctgtgtt
     1921 ccagaagtgt tggtaaacag cccacaaatg tcaacagcag aaacatacaa gctgtcagct
     1981 ttgcacaagg gcccaacacc ctgctcatca agaagcactg tggttgctgt gttagtaatg
     2041 tgcaaaacag gaggcacatt ttccccacct gtgtaggttc caaaatatct agtgttttca
     2101 tttttacttg gatcaggaac ccagcactcc actggataag cattatcctt atccaaaaca
     2161 gccttgtggt cagtgttcat ctgctgactg tcaactgtag cattttttgg ggttacagtt
     2221 tgagcaggat atttggtcct gtagtttgct aacacaccct gcagctccaa aggttcccca
     2281 ccaacagcaa aaaaatgaaa atttgaccct tgaatgggtt ttccagcacc attttcatga
     2341 gttttttgtg tccctgaatg caagtttaac atagcagtta ccccaataac ctcagtttta
     2401 acagtaacag cttcccacat caaaatattt ccacaggtta agtcctcatt taaagagctc
     2461 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     2521 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2581 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2641 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2701 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2761 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2821 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2881 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2941 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3001 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3061 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3121 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3181 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3241 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3301 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3361 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3421 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3481 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3541 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3601 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3661 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3721 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3781 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3841 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3901 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3961 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4021 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4081 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4141 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4201 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4261 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4321 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4381 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4441 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4501 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4561 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4621 acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
