LOCUS       sequence-489773-        4664 bp    DNA     circular SYN 20-OCT-2025
DEFINITION  In-Vitro Synthesis of mRNA encoding a Honeybee-optimized 
            mEmerald-labeled Nuclear Localization Signal (NLS) Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4664)
  TITLE     In-Vitro Synthesis of mRNA for Fluorescent Labeling of Honeybee 
            Embryos
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4664)
  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            "Unpublished"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-mEmerald(AM)
FEATURES             Location/Qualifiers
     source          1..4664
                     /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    1587..1721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1624..1643)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1678..1697
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2476..2496)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2476..2494)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2515..2531)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2515..2531)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2528..2550)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2539..2555
                     /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(2563..2569,2570..2587,2588..2593))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2608..2629
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2746..2763)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2917..3505)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2997..3016)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3676..4467,4468..4536))
                     /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     4299..4318
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4537..4641)
                     /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 gaattgttca
      781 ccggcgtggt gccaattttg gtggaattgg acggcgacgt gaacggccac aaattctcgg
      841 tgtcgggcga aggcgaaggc gacgccacct acggcaaatt gaccttgaaa ttcatttgta
      901 ccaccggcaa attgccagtg ccatggccaa ccttggtgac caccttgacc tacggcgtgc
      961 aatgtttcgc cagataccca gaccacatga aacaacacga cttcttcaaa tcggccatgc
     1021 cagaaggcta cgtgcaagaa agaaccattt tcttcaaaga cgacggcaac tacaaaacca
     1081 gagccgaagt gaaattcgaa ggcgacacct tggtgaacag gattgaattg aaaggcattg
     1141 acttcaaaga agatggcaac attttgggcc acaaattgga atacaactac aactcgcaca
     1201 aagtgtacat taccgccgac aaacaaaaga acggcattaa agtgaacttc aaaaccagac
     1261 acaacattga agatggctcg gtgcaattgg ccgaccacta ccaacaaaac accccaattg
     1321 gcgacggccc agtgttgttg ccagacaacc actacttgtc gacccaatcg aaattgtcga
     1381 aagacccaaa cgaaaagagg gaccacatgg tgttgttgga attcgtgacc gccgccggca
     1441 ttaccttggg catggacgaa ttgtacaaat aacctgcagg gatcataatc agccatacca
     1501 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     1561 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     1621 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     1681 gtttgtccaa actcatcaat gtatcttatc atgtctggat ccccaggaag ctcctctgtg
     1741 tcctcataaa ccctaacctc ctctacttga gaggacattc caatcatagg ctgcccatcc
     1801 accctctgtg tcctcctgtt aattaggtca cttaacaaaa aggaaattgg gtaggggttt
     1861 ttcacagacc gctttctaag ggtaatttta aaatatctgg gaagtccctt ccactgctgt
     1921 gttccagaag tgttggtaaa cagcccacaa atgtcaacag cagaaacata caagctgtca
     1981 gctttgcaca agggcccaac accctgctca tcaagaagca ctgtggttgc tgtgttagta
     2041 atgtgcaaaa caggaggcac attttcccca cctgtgtagg ttccaaaata tctagtgttt
     2101 tcatttttac ttggatcagg aacccagcac tccactggat aagcattatc cttatccaaa
     2161 acagccttgt ggtcagtgtt catctgctga ctgtcaactg tagcattttt tggggttaca
     2221 gtttgagcag gatatttggt cctgtagttt gctaacacac cctgcagctc caaaggttcc
     2281 ccaccaacag caaaaaaatg aaaatttgac ccttgaatgg gttttccagc accattttca
     2341 tgagtttttt gtgtccctga atgcaagttt aacatagcag ttaccccaat aacctcagtt
     2401 ttaacagtaa cagcttccca catcaaaata tttccacagg ttaagtcctc atttaaagag
     2461 ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     2521 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2581 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2641 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2701 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2761 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2821 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2881 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2941 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3001 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3061 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3121 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3181 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3241 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3301 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3361 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3421 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3481 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3541 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3601 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3661 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3721 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3781 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3841 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3901 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3961 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4021 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4081 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4141 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4201 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4261 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4321 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4381 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4441 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4501 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4561 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4621 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccac
//
