LOCUS       sequence-489766-        4655 bp    DNA     circular SYN 20-OCT-2025
DEFINITION  In-Vitro Synthesis of mRNA encoding a Honeybee-optimized 
            mCherry-labeled Nuclear Localization Signal (NLS) Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4655)
  TITLE     In-Vitro Synthesis of mRNA for Fluorescent Labeling of Honeybee 
            Embryos
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4655)
  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            "Unpublished"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-mCherry(AM)
FEATURES             Location/Qualifiers
     source          1..4655
                     /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    1578..1712
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1615..1634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1669..1688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2467..2487)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2467..2485)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2506..2522)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2506..2522)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2519..2541)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2530..2546
                     /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(2554..2560,2561..2578,2579..2584))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2599..2620
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2737..2754)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2908..3496)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2988..3007)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3667..4458,4459..4527))
                     /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     4290..4309
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4528..4632)
                     /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 cggccgcctc gaaaggcgaa gaagataaca
      781 tggccattat taaagaattc atgagattca aagtgcacat ggaaggctcg gtgaacggcc
      841 acgaattcga aattgaaggc gaaggcgaag gcagaccata cgaaggcacc caaaccgcca
      901 aactgaaagt gaccaaaggc ggcccactgc cattcgcctg ggacattctg tcgccacaat
      961 tcatgtacgg ctcgaaagcc tacgtgaaac acccagccga cattccagac tacctgaaac
     1021 tgtcgttccc agaaggcttc aaatgggaaa gagtgatgaa cttcgaagat ggcggcgtgg
     1081 tgaccgtgac ccaagactcg tcgctgcaag acggcgaatt catttacaaa gtgaaactga
     1141 gaggcaccaa cttcccatcg gacggcccag tgatgcaaaa gaaaaccatg ggctgggaag
     1201 cctcgtcgga aagaatgtac ccagaagatg gcgccctgaa aggcgaaatt aaacaaagac
     1261 tgaaactgaa agacggcggc cactacgacg ccgaagtgaa aaccacctac aaagccaaga
     1321 aaccagtgca actgccaggc gcctacaacg tgaacattaa actggacatt acctcgcaca
     1381 acgaagatta caccattgtg gaacaatacg aaagagccga aggcagacac tcgaccggcg
     1441 gcatggacga actgtacaaa taacctgcag ggatcataat cagccatacc acatttgtag
     1501 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     1561 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     1621 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     1681 aactcatcaa tgtatcttat catgtctgga tccccaggaa gctcctctgt gtcctcataa
     1741 accctaacct cctctacttg agaggacatt ccaatcatag gctgcccatc caccctctgt
     1801 gtcctcctgt taattaggtc acttaacaaa aaggaaattg ggtaggggtt tttcacagac
     1861 cgctttctaa gggtaatttt aaaatatctg ggaagtccct tccactgctg tgttccagaa
     1921 gtgttggtaa acagcccaca aatgtcaaca gcagaaacat acaagctgtc agctttgcac
     1981 aagggcccaa caccctgctc atcaagaagc actgtggttg ctgtgttagt aatgtgcaaa
     2041 acaggaggca cattttcccc acctgtgtag gttccaaaat atctagtgtt ttcattttta
     2101 cttggatcag gaacccagca ctccactgga taagcattat ccttatccaa aacagccttg
     2161 tggtcagtgt tcatctgctg actgtcaact gtagcatttt ttggggttac agtttgagca
     2221 ggatatttgg tcctgtagtt tgctaacaca ccctgcagct ccaaaggttc cccaccaaca
     2281 gcaaaaaaat gaaaatttga cccttgaatg ggttttccag caccattttc atgagttttt
     2341 tgtgtccctg aatgcaagtt taacatagca gttaccccaa taacctcagt tttaacagta
     2401 acagcttccc acatcaaaat atttccacag gttaagtcct catttaaaga gctccagctt
     2461 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     2521 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2581 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2641 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2701 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2761 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2821 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2881 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2941 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3001 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3061 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3121 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3181 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3241 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3301 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3361 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3421 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3481 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3541 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3601 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3661 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3721 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3781 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3841 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3901 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3961 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4021 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4081 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4141 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4201 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4261 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4321 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4381 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4441 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4501 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4561 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4621 aggggttccg cgcacatttc cccgaaaagt gccac
//
