LOCUS       sequence-360425-        6956 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses FLAG tagged FZD4 swapped with FZD6 CRDlinker domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6956)
  TITLE     HOLD - Choi lab 2022
REFERENCE   2  (bases 1 to 6956)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pcDNA FZD4_6CRDlinker
FEATURES             Location/Qualifiers
     source          1..6956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             494..511
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(534..551)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    540..764
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      810..1238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(897..916)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1107..1128
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1247..1267)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1252..1581
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1432..1567
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1494..1513
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1648..2442
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1702..1721)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2312..2331
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2618..2739
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2655..2674)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2709..2728
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2788..2804)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2788..2804)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2801..2823)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2812..2828
                     /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(2836..2842,2843..2860,2861..2866))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2881..2902
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3019..3036)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3190..3778)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3270..3289)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3949..4740,4741..4809))
                     /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     4572..4591
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4810..4914)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4989..5008)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5180..5559
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5560..5763
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5714..5734
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5808..5827
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5808..5826
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5860..5904
                     /codon_start=1
                     /product="leader sequence from mouse preprotrypsin"
                     /label=preprotrypsin leader
                     /translation="MSALLILALVGAAVA"
     CDS             5905..5928
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
ORIGIN
        1 gtgctatgtt gggaaccaaa acctagatgc cctcactggc tttgtggtgg ctcctctctt
       61 tacgtatttg gtgattggaa cgctgttcat tgcggcgggt ttggtggcct tattcaaaat
      121 tcggtccaat cttcaaaaag acgggacaaa gacagacaag ttggaaaggc taatggtcaa
      181 gatcggggtc ttctcagtac tgtacacggt tcctgcaacc tgtgtgattg cctgttattt
      241 ctatgaaatc tcaaactggg cactctttcg atattctgca gatgactcaa acatggcagt
      301 tgaaatgttg aaaattttta tgtctttgct cgtgggcatc acttcaggca tgtggatttg
      361 gtctgccaaa actcttcaca cgtggcaaaa gtgttctaac cgattggtga attctgggaa
      421 ggtaaagaga gagaagaggg ggaatggttg ggtgaagcca ggaaaaggca acgagactgt
      481 ggtatagctc gagcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac
      541 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
      601 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
      661 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
      721 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
      781 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
      841 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
      901 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
      961 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1021 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1081 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1141 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1201 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     1261 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     1321 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     1381 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     1441 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     1501 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     1561 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     1621 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     1681 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     1741 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     1801 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     1861 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     1921 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     1981 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     2041 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     2101 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     2161 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     2221 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     2281 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     2341 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     2401 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     2461 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     2521 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     2581 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     2641 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2701 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     2761 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2821 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2881 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2941 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3001 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3061 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3121 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3181 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3241 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3301 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3361 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3421 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3481 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3541 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3601 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3661 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3721 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3781 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3841 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3901 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3961 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4021 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4081 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4141 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4201 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4261 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4321 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4381 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4441 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4501 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4561 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4621 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4681 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4741 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4801 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4861 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4921 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     4981 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     5041 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     5101 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     5161 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     5221 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     5281 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     5341 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     5401 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     5461 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     5521 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     5581 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     5641 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     5701 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     5761 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
     5821 tatagggaga cccaagctgg ctagttaagc ttggtaccaa tgtctgcact tctgatccta
     5881 gctcttgttg gagctgcagt tgctgactac aaagacgatg acgacaagct tgcggccgca
     5941 cacagtctct tcacctgtga accaattact gttcccagat gtatgaaaat ggcctacaac
     6001 atgacgtttt tccctaatct gatgggtcat tatgaccaga gtattgccgc ggtggaaatg
     6061 gagcattttc ttcctctcgc aaatctggaa tgttcaccaa acattgaaac tttcctctgc
     6121 aaagcatttg taccaacctg catagaacaa attcatgtgg ttccaccttg tcgtaaactt
     6181 tgtgagaaag tatattctga ttgcaaaaaa ttaattgaca cttttgggat ccgatggcct
     6241 gaggagcttg aatgtgacag attacaatac tgtgatgaga ctgttcctgt aacttttgat
     6301 ccacacacag aatttcttgg tcctcagaag aaaacagaac aagtccaaag agacattgga
     6361 ttttggtgtc caaggcatct taagacttct gggggacaag gatataagtt tctgggaatt
     6421 gaccagtgtg cgcctccatg tggctacgat gctggcttgt acagccgctc agctaaggag
     6481 ttcacggata tttggatggc tgtgtgggcc agcctctgct tcatctccac caccttcacc
     6541 gtgctgacct tcctgattga ttcatccagg ttttcttacc ctgagcgccc catcatattt
     6601 ctcagtatgt gctataatat ttatagcatt gcttatattg ttcggctgac tgtaggccgg
     6661 gaaaggatat cctgtgattt tgaagaggcg gcagagcccg ttctcatcca agaaggactt
     6721 aagaacacag gatgtgcaat aattttcttg ctgatgtact tttttggaat ggccagctcc
     6781 atttggtggg ttattctgac actcacttgg tttttggcag ccggactcaa gtggggtcat
     6841 gaagccattg aaatgcacag ttcttatttc cacatcgcag cctgggctat tcccgcagtg
     6901 aaaaccattg tcatcttgat tatgagacta gtggatgccg atgaactgac tggctt
//
