LOCUS       sequence-373502-        4973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  vector backbone to clone insert downstream of Halo tag (Halo tag 
            codon optimized for Xenopus), for mRNA synthesis .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4973)
  AUTHORS   Dudley CE, van den Goor L, Miller AL
  TITLE     SNAP- and Halo-tagging and dye introduction protocol for live 
            microscopy in Xenopus embryos.
  JOURNAL   STAR Protoc. 2022 Aug 18;3(3):101622. doi: 
            10.1016/j.xpro.2022.101622. eCollection 2022 Sep 16.
  PUBMED    36035797
REFERENCE   2  (bases 1 to 4973)
  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: "STAR 
            Protoc."; date: "2022-08-18"; volume: "3(3):101622. doi"; pages: " 
            10.1016/j.xpro.2022.101622. eCollection 2022 Sep 16"
COMMENT     Sequence Label: pCS2+/C-Halo
FEATURES             Location/Qualifiers
     source          1..4973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        52..70
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     52..69
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             136..1023
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     polyA_signal    1042..1176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1066..1085)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1120..1139
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1289..1309)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1289..1307)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1328..1344)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1328..1344)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1341..1363)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1352..1368
                     /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(1376..1382,1383..1400,1401..1406))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1421..1442
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1559..1576)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1730..2318)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1810..1829)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2489..3280,3281..3349))
                     /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     3112..3131
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3350..3454)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3480..3935)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3617..3638)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3829..3848
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(4006..4973,1..17)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 atgctcgttt agggaaccgc cattctgcct ggggacgtcg gagcaagctt gatttaggtg
       61 acactataga atacaagcta cttgttcttt ttgcaggatc ccatcgattc gaattcaagg
      121 cctctcgagt ctagagcaga aatcggtact ggctttccat tcgaccccca ttatgtggaa
      181 gtcctgggcg agcgcatgca ctacgtcgat gttggtccac gcgatggcac ccctgtgctg
      241 ttcctgcacg gtaacccaac ctcctcctac gtgtggcgca acatcatccc acatgttgca
      301 ccaacccatc gctgcattgc tccagacctg atcggtatgg gcaaatccga caaaccagac
      361 ctgggttatt tcttcgacga ccacgtccgc ttcatggatg ccttcatcga agccctgggt
      421 ctggaagagg tcgtcctggt cattcacgac tggggctccg ctctgggttt ccactgggcc
      481 aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg agttcatccg ccctatccca
      541 acctgggacg aatggccaga atttgcccgc gagaccttcc aggccttccg caccaccgac
      601 gtcggccgca agctgatcat cgatcagaac gtttttatcg agggtacact gccaatgggt
      661 gtcgtccgcc cactgactga agtcgagatg gaccattacc gcgagccatt cctgaatcct
      721 gttgaccgcg agccactgtg gcgcttccca aacgagctgc caatcgccgg tgagccagct
      781 aacatcgtcg ctctggtcga agaatacatg gactggctgc accagtcccc tgtcccaaag
      841 ctgctgttct ggggcacccc aggcgttctg atcccaccag ccgaagccgc tcgcctggcc
      901 aaaagcctgc ctaactgcaa ggctgtggac atcggcccag gtctgaatct gctgcaagaa
      961 gacaacccag acctgatcgg cagcgagatc gctcgctggc tgagcacact cgagatttcc
     1021 ggctaatgta caaggttacg agatccagac atgataagat acattgatga gtttggacaa
     1081 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     1141 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     1201 atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcgcggccgc ggcgccaatg
     1261 cattgggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     1321 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     1381 tacgagccgg gagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     1441 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     1501 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     1561 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1621 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     1681 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1741 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     1801 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1861 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1921 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1981 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2041 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2101 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2161 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2221 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     2281 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2341 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2401 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2461 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2521 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2581 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     2641 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     2701 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     2761 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     2821 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2881 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2941 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3001 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3061 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3121 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3181 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     3241 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     3301 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     3361 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     3421 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     3481 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     3541 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3601 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     3661 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     3721 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     3781 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     3841 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     3901 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     3961 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagtcga ccgccaattc
     4021 aatatggcgt atatggactc atgccaattc aatatggtgg atctggacct gtgccaattc
     4081 aatatggcgt atatggactc gtgccaattc aatatggtgg atctggaccc cagccaattc
     4141 aatatggcgg acttggcacc atgccaattc aatatggcgg acctggcact gtgccaactg
     4201 gggaggggtc tacttggcac ggtgccaagt ttgaggaggg gtcttggccc tgtgccaagt
     4261 ccgccatatt gaattggcat ggtgccaata atggcggcca tattggctat atgccaggat
     4321 caatatatag gcaatatcca atatggccct atgccaatat ggctattggc caggttcaat
     4381 actatgtatt ggccctatgc catatagtat tccatatatg ggttttccta ttgacgtaga
     4441 tagcccctcc caatgggcgg tcccatatac catatatggg gcttcctaat accgcccata
     4501 gccactcccc cattgacgtc aatggtctct atatatggtc tttcctattg acgtcatatg
     4561 ggcggtccta ttgacgtata tggcgcctcc cccattgacg tcaattacgg taaatggccc
     4621 gcctggctca atgcccattg acgtcaatag gaccacccac cattgacgtc aatgggatgg
     4681 ctcattgccc attcatatcc gttctcacgc cccctattga cgtcaatgac ggtaaatggc
     4741 ccacttggca gtacatcaat atctattaat agtaacttgg caagtacatt actattggaa
     4801 gtacgccagg gtacattggc agtactccca ttgacgtcaa tggcggtaaa tggcccgcga
     4861 tggctgccaa gtacatcccc attgacgtca atggggaggg gcaatgacgc aaatgggcgt
     4921 tccattgacg taaatgggcg gtaggcgtgc ctaatgggag gtctatataa gca
//
