LOCUS       sequence-349341-        4374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for endogenous tagging of human GCH1 at the N-terminus 
            with halotag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4374)
  TITLE     iNDI donor vectors for endogenous tagging with Halotag
REFERENCE   2  (bases 1 to 4374)
  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"
COMMENT     Sequence Label: GCH1_Halo_N_allele
FEATURES             Location/Qualifiers
     source          1..4374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(23..40)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(194..782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(274..293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(953..1744,1745..1813))
                     /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     1576..1595
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1814..1918)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1986..2004
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2042..2064)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2164..2183
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2377..2399
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2391..2408
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2392..2408
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2815..3705
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     complement(4166..4182)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4166..4182)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4179..4201)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4190..4206
                     /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(4214..4220,4221..4238,4239..4244))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4259..4280
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
       61 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      121 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      181 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      241 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      301 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      361 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      421 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      481 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      541 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      601 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      661 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      721 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      781 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      841 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      901 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
      961 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     1021 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     1081 caatgatacc gcgagatcca cgctcaccgg ctccagattt atcagcaata aaccagccag
     1141 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     1201 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     1261 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     1321 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     1381 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     1441 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     1501 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     1561 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     1621 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     1681 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     1741 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     1801 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     1861 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     1921 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     1981 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     2041 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     2101 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     2161 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     2221 gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt
     2281 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     2341 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     2401 cggccagtga attcgcaata agtggagggg tcccgcctga tcctaggggc cggcgccctt
     2461 ttccttccct ccctgcgctt gcgaacccct ccgggcgtct ccggagccgc gacccctgct
     2521 ggggaccccg ggggcgggag gccgaggggc gcggcgaggg ccgtgacgcg aggcggggcc
     2581 ggccaatggg agcgctccgc ggcgcaggct gagccgccgg gccataaaaa ggaggcgcgg
     2641 ccgggctttc cagcctgtgg ccgctcccgg ctcggagtgt gatctaagca ggtcgcgtac
     2701 cttcctcagg tgactccggc cacagcccat tgtccgcggc caccggcgga gtttagccgc
     2761 agacctcgaa gcgccccggg gtccttcccg aacggcagcg gctgcggcgg gtccatggcc
     2821 gagatcggca ccggctttcc cttcgatcct cactacgtgg aagtgctggg cgagagaatg
     2881 cactatgtgg acgtgggccc cagagatgga acccctgtgc tgtttctgca cggcaaccct
     2941 accagcagct acgtgtggcg gaacatcatc cctcacgtgg cccctacaca cagatgtatc
     3001 gcccctgacc tgatcggcat gggcaagagc gataagcccg acctgggcta cttcttcgac
     3061 gaccacgtgc ggttcatgga cgcctttatt gaggccctcg gcctggaaga ggtggtgctg
     3121 gttattcacg attggggcag cgccctggga ttccactggg ccaagagaaa ccccgagaga
     3181 gtgaagggaa tcgccttcat ggaattcatc aggcccattc ctacctggga cgagtggcct
     3241 gagttcgcca gagagacatt ccaggccttc agaaccaccg acgtgggcag aaagctgatc
     3301 atcgaccaga acgtgttcat cgagggcacc ctgcctatgg gagtcgtcag accactgacc
     3361 gaggtggaaa tggaccacta cagagagccc tttctgaacc ccgtggaccg ggaacctctt
     3421 tggagattcc ccaacgagct gcccattgcc ggcgagcctg ctaatattgt ggccctggtg
     3481 gaagagtaca tggactggct gcatcagagc cccgtgccta agctgctgtt ttggggaaca
     3541 cccggcgtgc tgattcctcc tgctgaagct gccagactgg ccaagagcct gcctaattgc
     3601 aaggccgtgg atatcggccc tggcctgaat ctgctgcaag aggacaaccc cgatctgatc
     3661 ggctccgaga ttgccagatg gctgagcacc ctggaaatca gcggaggagg aggttctgga
     3721 ggcggtggaa gtggtggcgg aggtagcgag aagggccctg tgcgggcacc ggcggagaag
     3781 ccgcggggcg ccaggtgcag caatgggttc cccgagcggg atccgccgcg gcccgggccc
     3841 agcaggccgg cggagaagcc cccgcggccc gaggccaaga gcgcgcagcc cgcggacggc
     3901 tggaagggcg agcggccccg cagcgaggag gataacgagc tgaacctccc taacctggca
     3961 gccgcctact cgtccatcct gagctcgctg ggcgagaacc cccagcggca agggctgctc
     4021 aagacgccct ggagggcggc ctcggccatg cagttcttca ccaagggcta ccaggagacc
     4081 atctcaggtc agtgcgcccg cgggctgcta gagcgtgcgg gcggcggggg cggtgcttgc
     4141 aggaaacaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4201 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4261 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4321 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgc
//
