LOCUS       sequence-393064-        7984 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses human codon-optimized V. cholerae TnsA-TnsB fusion from a 
            CMV promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7984)
  AUTHORS   Lampe GD, King RT, Halpin-Healy TS, Klompe SE, Hogan MI, Vo PLH, 
            Tang S, Chavez A, Sternberg SH
  TITLE     Targeted DNA integration in human cells without double-strand breaks
            using CRISPR-associated transposases.
  JOURNAL   Nat Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1.
  PUBMED    36991112
REFERENCE   2  (bases 1 to 7984)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1."
COMMENT     Sequence Label: pSL2669 (pTnsABf_Vch)
FEATURES             Location/Qualifiers
     source          1..7984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(259..363)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(438..457)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        629..1008
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1009..1212
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1163..1183
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1257..1276
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1257..1275
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1284..1293
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2028..2048
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(3931..3949)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3932..3949)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3969..3986)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3975..4199
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4245..4673
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4332..4351)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4542..4563
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4682..4702)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4687..5016
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4867..5002
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4929..4948
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5083..5877
                     /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(5137..5156)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5747..5766
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6051..6172
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6088..6107)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6142..6161
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6221..6237)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6221..6237)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6234..6256)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6245..6261
                     /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(6269..6275,6276..6293,6294..6299))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6314..6335
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6452..6469)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6623..7211)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6703..6722)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7382..7984,1..189,190..258))
                     /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"
ORIGIN
        1 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
       61 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      121 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      181 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      241 gaaatgttga atactcatac tcttcctttt tcattattat tgaagcattt atcagggtta
      301 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      361 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
      421 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
      481 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
      541 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
      601 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      661 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      721 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      781 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      841 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      901 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      961 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     1021 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     1081 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     1141 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1201 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
     1261 acgactcact atagggagac ccagccacca tggccacatc cctgccaacc ccctctgcca
     1321 tcaccacaag cgccctggag tacgccttcc acacaccagc aaggaacctg accaagtccc
     1381 gcggcaagaa tatccacaga tacgtgagcg tgaagatgag caagaggatc acagtggagt
     1441 ccaccctgga gtgcgacgcc tgttaccact tcgattttga gccatctatc gtgcgcttct
     1501 gtgcccagcc catccggttt ctgtactatc tgaacggcca gagccactcc tatgtgcctg
     1561 acttcctggt gcagtttgat accaacgagt tcgtgctgta cgaggtgaaa agcgcctatg
     1621 ccaagaataa gccagacttt gatgtggagt gggaggccaa ggtgaaggca gcaacagagc
     1681 tgggcctgga gctggagctg gtggaggagt ccgacatccg cgataccgtg gtgctgaaca
     1741 atctgaagcg catgcaccgc tacgcctcca aggacgagct gaacaatgtg cacaactctc
     1801 tgctgaagat catcaagtat aatggagcac agtccgccag gtgcctggga gagcagctgg
     1861 gcctgaaggg aaggacagtg ctgccaatcc tgtgcgatct gctgtctagg tgtctgctgg
     1921 acacccgcct ggataagccc ctgtccctgg agtctcggtt cgagctggca agctacggag
     1981 gcagcggctc cggaaaacgg acagccgacg gaagcgagtt cgagtcacca aagaagaagc
     2041 ggaaagtcgg atccggctct ggaggcgcca agaagggctt cagctccttt caccgcaagg
     2101 ccgtgtctag ccaggacacc ctggagagca tcgagctggt gtcctctgcc aactgcctgg
     2161 agagcgtgac ataccaggac atctccgcct tccctgaaac catcgccgtg gagatcaatt
     2221 tcagactgag catcctgaga tttctggcca ggaagtgtga gacaatcgtg gccaagtcca
     2281 tcgagcctca ccgggtggag ctgcaacaga actatagccg gaagatccca agcgccatca
     2341 ccatctaccg ctggtggctg gcctttcgga aaagcgacta taatccaatc tccctggccc
     2401 ccaacatcaa ggatagaggc aatagggaga caaaggtgtc caccgtggtg gactctatca
     2461 tggagcaggc agtggagcgc gtgatcagcg gacggaaggt gaacgtgagt tccgcctata
     2521 agagagtgcg gagaaaggtg aggcagtaca atctgaccca cggcacaaag tacacctatc
     2581 ccaagtacga gtccgtgaga aagagggtga agaagaaaac ccctttcgag ctgctggcag
     2641 caggcaaggg agagagggtg gccaagcggg agtttaggcg catgggcaag aagatcctga
     2701 catctagcgt gctggagcgg gtggagatcg accacaccgt ggtggatctg ttcgccgtgc
     2761 acgaggagta tagaatccct ctgggcaggc cctggctgac ccagctggtg gactgctact
     2821 ctaaggccgt gatcggcttc tatctgggct ttgagccccc tagctacgtg tctgtgagcc
     2881 tggccctgaa gaacgccatc cagagaaagg acgatctgat ctcctcttat gagagcatcg
     2941 agaacgagtg gctgtgctac ggcatcccag acctgctggt gacagataat ggcaaggagt
     3001 tcctgagcaa ggcctttgac caggcctgtg agtccctgct gatcaacgtg caccagaata
     3061 aggtggagac acccgataat aagcctcacg tcgagaggaa ctacggcaca atcaatacca
     3121 gcctgctgga cgatctgccc ggcaagtcct tctctcagta cctccagagg gagggatatg
     3181 actccgtggg agaggccacc ctgacactga acgagatcag agagatctat ctgatctggc
     3241 tggtggatat ttaccacaag aagcccaatc agcgcggcac caactgccct aatgtggcct
     3301 ggaagaaggg ctgtcaggag tgggagcctg aagagtttag cggctccaag gacgagctgg
     3361 atttcaagtt tgccatcgtg gactacaagc agctgacaaa agtgggcatc accgtgtaca
     3421 aggagctgtc ttatagcaac gataggctgg cagagtacag gggcaagaag ggaaaccaca
     3481 aggtgcagtt caagtataat ccagagtgta tggccgtgat ctgggtgctg gacgaggata
     3541 tgaacgagta ctttacagtg aatgccatcg attacgagta tgcctctcgg gtgagcctgt
     3601 ggcagcacaa gtacaacatg aagtatcagg ccgagctgaa tagcgccgag tatgacgagg
     3661 ataaggagat cgacgccgag atcaagatcg aggagatcgc cgatagatcc atcgtgaaaa
     3721 ccaacaagat cagggcacgg agaaggggag caagacacca ggagaactct gccagggcca
     3781 agtccatctc taacgccaat cctgcctcta tccagaagca cgaggacgag atcgtgagcg
     3841 ccgataacga cgattgggac atcgattacg tgtaactgca gatatccatc acactggcgg
     3901 ccgctcgagc atgcatctag agggccctat tctatagtgt cacctaaatg ctagagctcg
     3961 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4021 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4081 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4141 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     4201 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     4261 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4321 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4381 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4441 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4501 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4561 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4621 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     4681 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4741 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     4801 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4861 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4921 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     4981 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     5041 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     5101 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     5161 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     5221 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     5281 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     5341 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     5401 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     5461 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     5521 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     5581 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     5641 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     5701 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     5761 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     5821 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     5881 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     5941 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     6001 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     6061 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     6121 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     6181 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     6241 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     6301 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     6361 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     6421 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     6481 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     6541 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6601 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6661 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6721 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6781 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6841 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6901 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6961 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     7021 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     7081 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     7141 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     7201 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     7261 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     7321 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     7381 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     7441 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     7501 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     7561 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7621 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     7681 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     7741 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     7801 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     7861 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7921 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7981 gctc
//
