LOCUS       sequence-233769-        7614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal SUMO tagged II-ShhN in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7614)
  AUTHORS   Zhang Y, Bulkley DP, Xin Y, Roberts KJ, Asarnow DE, Sharma A, Myers 
            BR, Cho W, Cheng Y, Beachy PA
  TITLE     Structural Basis for Cholesterol Transport-like Activity of the 
            Hedgehog Receptor Patched.
  JOURNAL   Cell. 2018 Nov 15;175(5):1352-1364.e14. doi: 
            10.1016/j.cell.2018.10.026. Epub 2018 Nov 8.
  PUBMED    30415841
REFERENCE   2  (bases 1 to 7614)
  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"; doi: 
            "10.1016/j.cell.2018.10.026"; journalName: "Cell"; date: 
            "2018-11-15- 15"; volume: "175"; issue: "5"; pages: "1352-1364.e14"
COMMENT     Sequence Label: pHTSHP-Smt-IIShhN
FEATURES             Location/Qualifiers
     source          1..7614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             81..1163
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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)."
     primer_bind     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1528..2628
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2602..2625
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2677..2688
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2698..2721
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2758..2781
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2788..2811
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2833..2856
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2860..3150
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(3707..3724)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3707..3723)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3716..3738)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3902..3919)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4072..4158
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4250..4277
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4296..4387
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4388..4456,4457..5248)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4606..4625)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5290..5803)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5485..5506)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5697..5716
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5914..6502
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6403..6422
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6656..6673
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6688..6828
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6754..6773)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6914..6936
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6930..7121)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagtgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcacttcacc aacaaggacc atagattatg aaaactgaag aaggtaaact ggtaatctgg
     1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2461 ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat cagcggccac
     2701 caccaccacc accaccacca cgactacgac atcccctcct ccgagaactc tgctagctgg
     2761 agccacccgc agttcgaaaa aagcggccac caccaccacc accaccacca cgactacgac
     2821 atcccctcct ccctggaagt tctgttccag gggcccggat ccgactcaga agtcaatcaa
     2881 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
     2941 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
     3001 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
     3061 ggtattagaa ttcaagctga tcagacccct gaagatttgg acatggagga taacgatatt
     3121 attgaggctc acagagaaca gattggtggg attatcgggc ccggcagggg gtttggaaag
     3181 aggcggcacc ccaaaaagct gaccccttta gcctacaagc agtttattcc caacgtagct
     3241 gagaagaccc taggggccag cggcagatat gaagggaaga tcacaagaaa ctccgaacga
     3301 tttaaggaac tcacccccaa ttacaacccc gacatcatat ttaaggatga ggaaaacacg
     3361 ggagcagacc ggctgatgac tcagaggtgc aaagacaagt taaatgcctt ggccatctct
     3421 gtgatgaacc agtggcctgg agtgaagctg cgagtgaccg agggctggga tgaggacggc
     3481 catcattcag aggagtctct acactatgag ggtcgagcag tggacatcac cacgtccgac
     3541 cgggaccgca gcaagtacgg catgctggct cgcctggctg tggaagcagg tttcgactgg
     3601 gtctactatg aatccaaagc tcacatccac tgttctgtga aagcagagaa ctccgtggcg
     3661 gccaaatgag cggccgcccg cgggcatgct tgaattcagg cttggcactg gccgtcgttt
     3721 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     3781 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     3841 tgcgcagcct gaatggcgaa tggcagcttg gctgttttgg cggatgagat aagattttca
     3901 gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg
     3961 gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg
     4021 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa
     4081 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     4141 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     4201 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc
     4261 ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct aaatacattc
     4321 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4381 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4441 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4501 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4561 tcgccccgaa gaacgtttcc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4621 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4681 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4741 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4801 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4861 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4921 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4981 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5041 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5101 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5161 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5221 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5281 gattgattta ccccggttga taatcagaaa agccccaaaa acaggaagat tgtataagca
     5341 aatatttaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5401 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5461 cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5521 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5581 tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5641 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5701 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5761 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtaaaagga tctaggtgaa
     5821 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5881 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5941 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6001 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6061 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6121 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6181 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6241 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6301 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6361 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6421 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6481 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6541 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6601 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     6661 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     6721 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6781 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     6841 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     6901 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     6961 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgca gcgattcaca gatgtctgcc
     7021 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     7081 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     7141 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     7201 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     7261 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     7321 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     7381 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     7441 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     7501 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     7561 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga aatt
//
